Heterologous Expression of Pteris vittata Phosphate Transporter PvPht1

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Environmental Processes

Heterologous Expression of Pteris vittata Phosphate Transporter PvPht1;3 Enhances Arsenic Translocation to and Accumulation in Tobacco Shoots Yue Cao, Hua-Yuan Feng, Dan Sun, Guohua Xu, Bala Rathinasabapathi, Yanshan Chen, and Lena Q. Ma Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b02082 • Publication Date (Web): 14 Aug 2019 Downloaded from pubs.acs.org on August 15, 2019

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Environmental Science & Technology

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Heterologous Expression of Pteris vittata Phosphate Transporter PvPht1;3 Enhances Arsenic Translocation to and Accumulation in Tobacco Shoots

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Yue Cao,† Huayuan Feng,† Dan Sun,†§ Guohua Xu,‡ Bala Rathinasabapathi§, Yanshan Chen,†,∥*, 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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§Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611, United

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States

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∥School of the Environment, Nanjing Normal University, Nanjing, Jiangsu 210023, 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; E-mail addresses:

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[email protected] and [email protected]

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ACS Paragon Plus Environment


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TOC

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ABSTRACT

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Arsenic-hyperaccumulator Pteris vittata is efficient in As accumulation and has been used

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in phytoremediation of As-contaminated soils. Arsenate (AsV) is the predominant As species

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in aerobic soils and is taken up by plants via phosphate transporters (Pht) including P. vittata.

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In this work, we cloned the PvPht1;3 full length coding sequence from P. vittata and

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investigated its role in As accumulation by yeast and plants. PvPht1;3 complemented a yeast P

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uptake mutant strain and showed a stronger affinity and transport capacity to AsV than

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PvPht1;2. In transgenic tobacco, PvPht1;3 enhanced AsV absorption and translocation,

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increasing As accumulation in the shoots under both hydroponic and soil experiments. Based

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on the expression patterns via qRT-PCR, PvPht1;3 was strongly induced by P deficiency, but

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not As exposure. To further understand its expression pattern, transgenic Arabidopsis thaliana

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and soybean expressing the GUS reporter gene, driven by PvPht1;3 promoter, were produced.

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The GUS staining showed that the reporter gene was mainly expressed in the stele cells,

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indicating that PvPht1;3 was expressed in stele cells and was likely involved in P/As

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translocation. Taken together, the data suggested that PvPht1;3 was a high-affinity AsV

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transporter and was probably responsible for efficient As translocation in P. vittata. Our results

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suggest that expressing PvPht1;3 enhances As translocation and accumulation in plants,

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thereby improving phytoremediation of As-contaminated soils.

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INTRODUCTION

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Arsenic (As) is a highly toxic metalloid, which is a carcinogen based on the Agency for

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Toxic Substances & Disease Registry in the US. It is present in soils as both organic and

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inorganic forms, with inorganic As being more dominant and toxic. Chronic exposure to As-

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contaminated soils and consumption of contaminated food crops are the major exposure

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pathways to humans.1 Therefore, it is important to reduce As levels in soils to reduce As risks.

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Phytoextraction, one of the phytoremediation technologies, is based on hyperaccumulating

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plants to remove As from soils, which has been used to clean up As-contaminated soils.2, 3

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Arsenate (AsV) is the dominant As species in aerobic soils and is taken up by phosphate

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(P) transporters by plants including As-hyperaccumulator P. vittata.4,5 Asher et al. found that

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P in growth medium inhibits AsV absorption by barley seedlings, suggesting that AsV

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absorption by plants might be related to P uptake pathways.6 Later, researchers also found

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similar phenomena in other plants including velvet grass (Holcus lanatus), Arabidopsis

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thaliana, rice (Oryza sativa), and wheat (Triticum aestivum).7-10 The data suggest that both P

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and AsV enter plant roots through P transporters. Genetic studies of P transporter 1 (Pht1), a

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subfamily of Pht superfamily, have provided direct evidence that Pht mediates both AsV and

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P acquisition.11-15 In Arabidopsis, AsV is taken up via AtPht1;1 and AtPht1;4, which exhibit

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different affinities to AsV and their deletion increases AsV tolerance.11, 16 In rice, P transporters

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OsPht1;4 and OsPht1;8 mediate AsV uptake.17,18 It was reported that deleting OsPht1;4

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decreases As accumulation by 16-32% in rice after AsV exposure, thereby increasing its

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tolerance to AsV exposure.17 Besides, OsPht1;8 also plays an important role in AsV uptake

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and translocation in rice,18 with its overexpressing enhancing AsV accumulation in rice.9

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Besides OsPht1;4 and OsPht1;8, OsPht1;1 may also be involved in AsV uptake and its

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modulation affects As accumulation in rice.19 Expression patterns of PTs in tobacco (Nicotiana

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tabacum) have been characterized, P-starvation enhanced the NtPht1;1 expression in both the

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leaves and roots, but P-depletion induction of NtPht1;2 only occurred in the roots.20

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As the first-known As-hyperaccumulator, P. vittata (Chinese brake fern) is efficient in As uptake

and

translocation,

exhibiting

markedly

higher

As

uptake

than

non4


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hyperaccumulators.21-24 Similar to other plants, increase in P content in growth media inhibits

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AsV assimilation by P. vittata,22,25 suggesting that P uptake system also plays a key role in its

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efficient AsV accumulation. The data confirmed that AsV uptake and transport in P. vittata are

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also mediated by P transporters.

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Currently, three P. vittata Phts, including PvPht1;1, PvPht1;2 and PvPht1;3, have been

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identified.26 PvPht1;1 and PvPht1;2 share 99% identity with only 8 amino acid residues be

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different between them, hence they may have overlapping functions. PvPht1;1/2 and PvPht1;3

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localize to the plasma membrane and are induced under P starvation. When heterologous

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expressed in yeast and plants, PvPht1;2 showed high efficiency in P acquisition but low

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capacity to AsV transport.26,

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utilization in transgenic tobacco plants.27 PvPht1;3 also showed a high affinity to transport P

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in yeast. However, different from PvPht1;2, PvPht1;3 is also an efficient AsV-transporter in

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yeast, so its expression increases As accumulation in yeast cells, making them sensitive to

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AsV.25

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Therefore, heterologously expressed PvPht1;2 enhances P

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Though PvPht1;3 has been studied in yeast, its role in plants has not been tested. In this

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study, we cloned PvPht1;3 cDNA and its promoter sequence. Using a yeast P uptake mutant

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strain, we identified PvPht1;3 As/P transport capacity. To study its functions in AsV uptake

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and translocation in plants, we transformed PvPht1;3 or PvPht1;3 promoter into model plant

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tobacco or Arabidopsis thaliana. We measured As transport and translocation of transgenic

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lines and wild-type as well as expression pattern of PvPht1;3. This study demonstrated the

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plant functions of the key AsV-transporter PvPht1;3, suggesting that transgenic approach based

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on PvPht1;3 can be used to enhance As accumulation in plants for more efficient

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phytoremediation.

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MATERIALS AND METHODS

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Growth of P. vittata. Spores of P. vittata were collected from Florida, USA, which were sowed on potting soils at a greenhouse at Nanjing University21. To keep soil moist, potting

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soils were covered with transparent plastic films. After 2 months of growth, sporophytes were

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transplanted into separate pots following Fu et al.28 For transcript analysis, the sporophytes 5



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with 4 fronds were cultured in 0.2 strength (0.2X) Hoagland nutrient solution (HNS), which

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contained 100 μM KH2PO4 (+P), 0 μM KH2PO4 (−P), or 100 μM KH2PO4/50 μM

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Na2HAsO4·7H2O (+As) for 3 days.

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Total RNA Preparation and Quantitative RT-PCR Analysis in P. vittata. The roots

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and fronds of P. vittata were cleaned by deionized water and then ground in a mortar and

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pestle with liquid nitrogen. Total RNA was extracted from P. vittata using Plant Total RNA

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Kit (Sigma-Aldrich, USA). First-strand cDNA was synthesized via reverse transcription

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using HiScript II One Step RT-PCR Kit (Vazyme Biotech, Nanjing, China). The qRT-PCR

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analysis was conducted using the CFX Connect Real-Time PCR Detection System (BIO-

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RAD), with the products being labeled using SYBR Green PCR Master Mix (Vazyme

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Biotech, Nanjing, China) and P. vittata actin gene (PvActin) being used as an internal

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control.29 Relative expression levels of PvPht1;3 (Accession No. KM192137) were analyzed

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by 2−ΔΔCT method of relative quantification. Gene-specific primers used for qRT-PCR

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included PvPht1;3 5′-TTC ACC GCA ATC GTC ATC GC -3′ and 5′- GGG AGG GTT CCA

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GGT TTG TCT -3′ and PvActin 5′-GGG CAG TAT TTC CAA GCA TAG TGG G-3′ and 5′-

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TGC CTC GCT TTG ATT GAG CCT CAT C-3′.

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Functional Complementation Assay of PvPht1;3 in a Yeast Mutant Strain

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Defective in P Uptake. PvPht1;3 coding sequences were first subcloned from cDNA of P.

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vittata into the cloning TOPO vector (TSING KE, Nanjing, China) using primers 5′- ATG

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GCT GGG GGA CAG CAG -3′ and 5′- TTA CAG TTG ATC GTT ACC ATC CGG -3′.

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Adapters were added to PvPht1;3 CDS using primers 5′-tcg acg gat tct aga act agt ATG GCT

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GGG GGA CAG CAG -3′ and 5′- gtc gac ggt atc gat aag ctt TTA CAG TTG ATC GTT ACC

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ATC CGG -3′(underline represents recombinant sequence). The PCR product was then

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cloned into the GLU1 promoter cassette of pAG426GLU-ccdB (Addgene,

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http://www.addgene.org/) using the Trelief SoSoo Cloning Kit (TSING KE, Nanjing, China).

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The methods for yeast transformations followed the high-efficient yeast transformation by

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Gietz et al.30

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The above constructs, along with empty vector, were transformed into the yeast mutant 6


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strain EY917 (lacking five P transporters PHO84, PHO87, PHO89, PHO90, and PHO91),

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which is defective in P uptake.31 To maintain the growth, EY917 mutant strain harbors a

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construct with PHO84, whose expression is induced by galactose in medium but inhibited by

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glucose. Based on this yeast system, PvPht1;2 or PvPht1;3 was transformed into EY917

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mutant strain with pAG426GLU-ccdB vector. Therefore, the expression of PvPht1;2 or

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PvPht1;3 was induced by glucose in the medium. Transformed yeast was grown on synthetic

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dropout (-Trp/-Ura) medium containing normal P concentration (7 mM) and 100 mM

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galactose, pH 5.8 for 4 d. Afterward, positive transformants were incubated in a shaker to an

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optical density OD600 of 1. Cells were then harvested by centrifugation at 3000 rpm for 10

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min, washed with sterilized water three times, and adjusted to an OD600 of 1.0. Aliquots of 5

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µL of 10-fold serial gradient dilutions were spotted on agar plates with or without 0.5 mM

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AsV and incubated at 30°C for 4 d.32

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Analysis of Yeast Growth and AsV Tolerance. PvPht1;2 and PvPht1;3 yeast

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transformats were cultured in a synthetic defined (SD) medium, which contained 0.67% yeast

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nitrogen base (without amino acids), 2% (w/v) glucose or 2% (w/v) galactose (induction

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medium), and 1.92 g L-1 yeast synthetic dropout (Sigma-Aldrich, USA), pH 5.8. To test their

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tolerance to As, PvPht1;2 and PvPht1;3 yeast transformats were cultured in a liquid SD

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medium with 2% [w/v] galactose to reach desired OD600, which was then centrifugated and

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diluted with sterile water. For drop assays, yeast cells were grow on SD plates (with 2% [w/v]

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glucose) containing 0.5 mM AsV for ΔEY917 expressing PvPht1;3 or PvPht1;2.

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Plant Expression Vector Construction and Transgenic Plant Generation and

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Selection. The PvPht1;3 CDS from P. vittata cDNAs used primers 5′-acg ggg gac tct aga gga

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tcc ATG GCT GGG GGA CAG CAG -3′ and 5′-ggg aaa ttc gag ctc ggt acc TTA CAG TTG

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ATC GTT ACC ATC CGG -3′ (underline to represent the recombinant sequence). The PCR

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product was recombined to the 35S promoter cassette of pSN1301 (pCAMBIA1301,

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CAMBIA), which used the Trelief SoSoo Cloning Kit (Tsing Ke, Nanjing, China). The

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constructed binary vector (named pSN1301-PvPht1;3) was then transformed into

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Agrobacterium strain C58 by electroporation. Transformation of transgenic tobacco plants

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was carried out using the Agrobacterium transformants.33, 34 Based on hygromycin resistance 7



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and GUS staining, PvPht1;3 expressing transgenic positive lines were identified. PvPht1;3 promoter sequence was cloned using primers 5′-cag gtc gac tct aga gga tcc

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CGA CGG CAG TGT CTG AGG CT -3′ and 5′- cct cag atc tac cat ggt acc CCA ATG CAC

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CTC GTC GTA -3′ (underlining indicates recombination sequences). The vector pSN1300

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(pCAMBIA1300, CAMBIA) was digested with two restriction enzymes, BamHI and KpnI.

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The PvPht1;3 promoter PCR product was inserted into the vector after digestion by

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recombination. After this step, the Cauliflower mosaic virus (CaMV) 35S promoter was

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replaced by the PvPht1;3 promoter in front of the GUS reporter gene. The newly-derived

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binary vector was transformed into Agrobacterium tumefaciens strain GV3101 for

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transformation of Arabidopsis thaliana, or introducyion into A. rhizogenes strain K599 for

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transformation of soybean hairy roots.35

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Agrobacterium-mediated transformation of A. thaliana was performed using floral dip

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transformation method.36 The healthy A. thaliana contained ~20–30 inflorescences and some

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maturing siliques were chosen for transformation experiments. The floral-dip liquid medium

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was composed of 2 ng μL-1 6-BA, 1% B5 (200 ×) basal medium, 100 μg μL-1 sucrose, 350 μL

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surfactant Silwet L-77, 1 M sodium hydroxide and Agrobacterium GV3101 (OD600: 1.0).37

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Place the plants upside down and dip their plantlets in floral-dip liquid medium for 5 min.

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The plants were then covered with plastic wrap to maintain high humidity. After dark culture

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for 24 h, the infected A. thaliana plantlets were moved to normal incubator and propagated

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until seeds were collected. The transgenic seeds were germinated on 1/2 MS containing

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kanamycin (100 mg L–1) and the positive lines were used for GUS staining to investigate the

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tissue cell location of PvPht1;3.

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The transformation of soybean (Glycine max) hair roots was also carried out.38 First, the

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soybean cotyledonary nodes were wounded with a scalpel, which was previously dipped into

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the cultures of A. rhizogenes strain K599 to produce transgenic hairy roots. Then the plants

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consisting of a wild-type shoot with transgenic hairy roots were transformed using the

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hypocotyl injection method. The transgenic soybean hairy roots were selected by adding 100

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mg L–1 kanamycin in the culture medium to be screened through PCR assay. Selected 8


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transgenic soybean composite plants were used for further GUS staining. Quantitative RT-PCR Analysis of Transgenic Tobacco. The tobacco seedlings were

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cleaned by deionized water and then ground in a mortar and pestle with liquid nitrogen. Total

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RNA was extracted from using Plant Total RNA Kit (Sigma-Aldrich, USA). The synthesis of

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the first-strand cDNA was conducted using HiScript II 1-Step RT-PCR Kit (Vazyme Biotech,

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Nanjing, China). Tobacco actin gene was used as a control based on LeActin primers 5′-TTC

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CGT TGC CCA GAG GTC CT-3′ and 5′-GGG AGC CAA GGC AGT GAT TTC-3′.

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PvPht1;3 expression-specific primers used for qRT-PCR included 5′-TTC ACC GCA ATC

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GTC ATC GC -3′ and 5′- GGG AGG GTT CCA GGT TTG TCT -3′.

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Histochemical Localization of GUS Expression. For the histochemical analysis 39, the

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transgenic soybean roots and transgenic A. thaliana tissue samples were submerged in GUS

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reaction mix [0.05 mM sodium phosphate buffer, 1 mM X-gluc and 0.1% (v/v) Triton X-100,

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pH 7.0], and were incubated at 37℃ overnight. Green tissues were distained with ethanol

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prior to observation. The stained tissues were photographed using an OLYMPUS MVX10

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steromicroscope, with a color CCD camera (Olympus, USA). The hand-held sections were

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transferred onto a slide and visualized under an Olympus BX51T stereomicroscope, with a

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color CCD camera (Olympus, USA).

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Growth of Transgenic Tobacco. In hydroponic experiments, transgenic tobacco and

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wild-type (WT) seeds were germinated on media containing 1/2 Murashige and Skoog (MS)

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salts, 1% sucrose and 0.5 g L-1 MES at pH 5.9, and solidified with 0.8% agar. After

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synchronize germination in the dark at 4°C for 2 d, the plates were placed in a growth

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chamber at 25°C with a 16-h light/8-h dark period. Uniform 2-week old tobacco seedlings

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were transferred to 0.2X HNS and grown for 21 d. For As accumulation, seedlings were

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exposed to 20 μM AsV for 1 d. In soil experiment, As-contaminated soil was obtained from a

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farmland in Qixiashan, Nanjing, China, with As concentration of 53 mg kg-1. The soils were

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collected from the top 20 cm, air-dried, and passed through 2 mm sieve. The soil properties

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are as follows: total P: 1.09 g kg-1; total As: 53 mg kg-1; available P: 13.2 mg kg-1; available

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As: 9.66 mg kg-1; total K: 8.80 g kg-1; total Fe: 28.2 g kg-1; and total Mn: 1.87 g kg-1. For 9



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available As determination, 1 g soil was extracted with 25 mL 0.05 M NH4H2PO4 solution for

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16 h (25℃, 250 rpm/min). The samples were then centrifuged at 3500 rpm for 15 min and the

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supernatants were filtered with 0.45 μm filters. 40As concentrations in the filtrates were then

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determined using ICP-MS. The air-dried soil (1.5 kg) was weighed into each pot, uniform 2-

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week old tobacco seedlings were grown in pots for 30 d. All plants were watered throughout

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the study to keep the soil at ~60% of field capacity. After harvest, plant dry biomass and As

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concentrations were determined.

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Arsenic Determination in Tobaccos and Arsenic Speciation in the Xylem Sap. Fresh

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plant biomass was lyophilized (FreezZone 12, LABCONCO) and stored at −80°C. Following

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USEPA Method 3050B, total As in plant samples were first digested with 50% HNO3 at

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105°C, which was then determined using inductively coupled plasma mass spectrometry

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

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After 21 d of cultivation in 0.2X HNS, the transgenic and WT tobacco seedlings were

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exposed to 20 μM AsV for 1 d. The stems were then cut at 2 cm above the roots, with the cut

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surfaces being were cleaned by deionized water. The xylem sap was collected by pipet from

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the cut surface for 2 h. After filtering through 0.22 μm filters, the samples were diluted for As

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speciation determination using high performance liquid chromatography (HPLC; Waters

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2695) coupled with ICP-MS.41

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Quality Assurance and Statistical Analysis. For quality assurance, we used indium as

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an internal standard, which was added into samples, calibration standards, and blanks. In

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addition, we used certified reference material (GSB 21, Chinese geological reference

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materials) as a control for transgenic and WT tobacco samples for quality assurance, which

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were within expected values.

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All data are analyzed by SPSS 20.0 software. The significant differences were

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determined as the mean of 3-6 replicates with standard error followed by Tukey’s mean

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group tests at p < 0.05.

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RESULTS AND DISCUSSION

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PvPht1;3 efficiently Transported AsV in a Yeast Mutant Strain Lacking P Transporters

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In a previous study, PvPht1;3 was investigated in yeast mutant strain lacking two P

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transporters.26 To understand the functions of PvPht1;3 and PvPht1;2 in P and AsV transport

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in yeast, a mutant strain EY917 lacking five P transporters (PHO84, PHO87, PHO89, PHO90,

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and PHO91) was used.31, 42 Maintaining its growth, EY917 harbors a construct where the yeast

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P transporter gene PHO84 is driven by the Gal promoter, with its expression being induced by

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galactose but inhibited by glucose. While the mutant strain growth can be restored using

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galactose as the C source, PHO84 expression is repressed using glucose as the C source, so the

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yeast cannot survive. Based on this yeast system, we transformed PvPht1;2 or PvPht1;3 into

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yeast, driven by the glucose promoter in pAG426GLU-ccdB vector. Hence, the expression of

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PvPht1;2 and PvPht1;3 can be induced by glucose and inhibited by galactose in EY917,

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opposite with the PHO84 in yeast mutant strain. Under the medium with galactose, all three

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transformants maintained P uptake by PHO84. Same growth phenotype showed that the growth

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rate of different transformants were similar in the initial stage of test (Figure 1A). When glucose

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was used, EY917 strain transformed with empty vector was unable to grow due to the inhibition

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of PHO84. In this case, PvPht1;2 and PvPht1;3 transformants can both complement the growth

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of yeast mutant strain (Figure 1A). After exposure to 0.5 mM AsV, EY917 strain transformed

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with PvPht1;3 showed stronger inhibition by AsV than EY917 transformed with PvPht1;2

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(Figure 1A).

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Furthermore, using turbidimetry at A600, we monitored yeast growth of PvPht1;2 and

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PvPht1;3 transformants under medium containing 1.5 mM P, 500 µM AsV (+AsV) or 0 µM

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AsV (-AsV) (Figure 1BC). The growth rate coefficients were determined via regression of the

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logarithmic growth phase. Without AsV, PvPht1;3 transformants showed 6-fold better growth

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than PvPht1;2 transformants. Better growth of PvPht1;3 transformants indicated that PvPht1;3

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was more efficient to transport P into yeast cells than PvPht1;2. Under AsV exposure, yeast

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cells expressing PvPht1;3 showed prominent growth inhibition (Figure 1C). In contrast, EY917

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expressing PvPht1;2 exhibited better AsV tolerance than EY917 expressing PvPht1;3 (Figure

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1B), suggesting that PvPht1;3 had stronger AsV transport capacity than PvPht1;2.

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To determine the kinetic properties of PvPht1;3, AsV uptake experiment was performed

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under 1-50 μM AsV and 1.5 mM P using the transformed yeast mutant strain. Data showed

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that PvPht1;3 mediated AsV uptake followed Michaelis-Menten kinetics, exhibiting an

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apparent Km of 5.84 μM (Figure 1D). The data suggested that PvPht1;3 had higher AsV

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affinity than its homolog PvPht1;2 (Km=13 μM), which showed little ability to transport

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AsV.27,26 To further compare their AsV uptake capacity, AsV concentrations in yeast mutant

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cells were determined. After growing in medium with 1.5 mM P + 150 µM AsV for 3 h, the

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PvPht1;3-expressing cells accumulated 2.4-fold more As than cells expressing PvPht1;2

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(Figure 1E). The data clearly showed that PvPht1;3 had higher AsV transport capacity than

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PvPht1;2.

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Using yeast strain PAM defective in P uptake, DiTusa et al also showed higher AsV

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transport capacity of PvPht1;3 than PvPht1;1/2 and AtPht1;5.26 Moreover, its affinity to P or

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AsV was comparable. Hence, PvPht1;3 was an efficient AsV-transporter.26 While the PAM

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yeast lost two P-transporters (PHO84 and PHO89),43 EY917 used in this study was defective

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in five P-transporters, thereby almost losing its P/AsV uptake function.31 Lacking all yeast P

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transporters, the P/AsV uptake by EY917 strain completely depended on the expressed target

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protein. Therefore, EY917 strain is more accurate to characterize the P/AsV transport capacity

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of target P transporters. Consistent with previous results, our study confirmed that PvPht1;3

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had strong AsV transport capacity.

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Heterologous Expressing PvPht1;3 Increased As Translocation in Tobacco Plants.

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Though PvPht1;3 has been studied in yeast, its plant functions are still unclear. P. vittata

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transformation is difficult, with the stable transformation efficiency of P. vittata gametophytes

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being only 0.012%.44 Thus, to further understand its plant functions, we generated PvPht1;3

293

transgenic tobacco lines (PvPht1;3-Ex) using the constitutive CaMV35S promoter. Three

294

independent transgenic T2 lines (Ex1-Ex3) were selected based on transgene expression

295

verified by qRT-PCR, in which PvPht1;3 transcripts were strongly expressed (Figure 2A). The

296

PvPht1;3 lines and WT plants were cultured in 0.2X HNS with 100 µM P, with all three

297

transgenic plants growing similarly as WT plants. The shoot P concentrations of Ex1-Ex3 were 12


Page 13 of 26


298

8.8-30% higher than those of WT plants, with root P showing little difference (Figure 2B),

299

indicating that expressing PvPht1;3 promoted P accumulation in the shoots of transgenic lines.

300

Under 20 μM AsV treatment for 1 d, the As concentrations in the shoots were 103-196% higher

301

than that of WT plants (Figure 2D). However, the As concentration in the roots of transgenic

302

lines were comparable with WT plants, resulting in 2.2-4.7 fold higher As translocation to the

303

shoots (Figure 2C-E).

304

To further determine AsV uptake of PvPht1;3-exprssing lines, their uptake kinetics was

305

investigated using 1-50 μM AsV. Without P, the AsV uptake kinetics followed the Michaelis-

306

Menten equation (Table 1). Maximum uptake velocity (Vmax) of AsV in PvPht1;3 lines was

307

40-84% higher than WT plants, with the Km of the transgenic lines being similar to WT plants.

308

With P, AsV uptake was suppressed in both transgenic and WT lines (Figure 3A, Table 1).

309

Under 100 μM P, AsV uptake was linear, showing that P strongly competed with AsV, with

310

the slope being 4-fold greater in PvPht1;3 lines than WT plants (Table 1). The data indicated

311

that PvPht1;3 promoted AsV uptake in transgenic tobacco plants. To further understand the

312

higher As translocation in PvPht1;3 lines, As speciation in the xylem sap was determined after

313

exposing to 20 μM AsV for 1 d. In the xylems of both lines, AsV was the predominant species,

314

with AsV concentrations being 47-106% higher in Ex1-Ex3 lines than WT plants (Figure 3B).

315

In contrast, AsIII concentrations in the xylems of both lines were comparable (Figure 3B),

316

suggesting that PvPht1;3 enhanced AsV translocation in xylem, probably by mediating AsV

317

transport into root xylem.

318

In our previous work, expressing PvPht1;2 enhanced P uptake and translocation

319

significantly. However, As accumulation in PvPht1;2 transgenic lines was comparable to that

320

of WT tobaccos in both hydroponic solution and As-contaminated soil.27 Unlike PvPht1;2,

321

expressing PvPht1;3 increased AsV uptake and translocation to the shoots (Figures 3 and 4),

322

but showing slight effect on P concentrations in transgenic tobacco (Figure 2B).

323

PvPht1;3 Expression Enhanced As Accumulation in Tobacco in Soil Experiment.

324

Since PvPht1;3 increased As accumulation in transgenic tobacco under hydroponic 13



Page 14 of 26

325

system, it may be an ideal candidate gene to enhance shoot As accumulation to improve

326

phytoremediation. For this purpose, PvPht1;3-expressing tobacco and WT plants were grown

327

in a contaminated soil with 53 mg kg-1 As for 30 d. Two transgenic tobacco lines grew similarly

328

as WT plants (Figure 4A). Consistent with hydroponic experiment, transgenic tobacco

329

translocated more As from the roots to shoots, accumulating 1.08-1.23 and 0.77-0.89 mg kg-1

330

As in the leaves and stems, being 42-62%, and 57-81% higher than WT plants (Figure 4B).

331

However, the As concentrations in the roots showed little difference from WT plants. The

332

results were consistent with hydroponic experiments, suggesting that PvPht1;3 enhanced As

333

translocation and accumulation in plants.

334

The As content in plants overexpressing Pht1 genes have been measured by others.

335

OsPht1;1, OsPht1;4 and OsPht1;8 made different contributions to As uptake and accumulation

336

in rice, with their overexpression increasing As accumulation in rice shoots under hydroponic

337

systems.19,45,46 In A. thaliana, mutants of AtPht1;1 and AtPht1;5 showed increased tolerance to

338

AsV, but no change in As accumulation was observed when AtPht1;1 or AtPht1;5

339

overexpression lines were compared to WT plants.11, 13 In fact, few results showed that Pht1

340

expression in plants enhances As uptake from As-contaminated soil. However, with its strong

341

AsV transport capacity, PvPht1;3 expression significantly promoted As accumulation in

342

transgenic plants under both hydroponic and soil systems. The data based on PvPht1;3 are

343

different from other P transporters mediating AsV transport in plants, though the mechanisms

344

are still unclear.

345

PvPht1;3 Exhibited High Expression in Plant Root Stele

346

It is likely that P. vittata takes up AsV from soils via P transporters.22 PvPht1;3 transports

347

AsV in yeast system and transgenic plants, thereby playing a crucial role in AsV uptake and

348

transport in P. vittata. To further understand its role in P. vittata, its expression patterns were

349

investigated using semi-quantitative reverse RT-PCR. PvPht1;3 was abundantly expressed in

350

both the roots and fronds (Figure 5A). To further confirm the results, quantitative RT-PCR was

351

performed with PvActin as a control, showing comparable PvPht1;3 transcript levels in the

352

roots and fronds (Figure 5B). Furthermore, PvPht1;3 expression was increased by 2.5-fold 14


Page 15 of 26


353

responding to P deficiency. However, it was not affected by AsV exposure under different P

354

regimes (Figure 5C), indicating that PvPht1;3 was relatively stable under As exposure.

355

To acquire P from environment, plant P transporters are induced by P starvation.47,48

356

Similarly, PvPht1;3 transcripts were up-regulated under P deficiency (Figure 5C), suggesting

357

that PvPht1;3 may participate in P uptake of P. vittata. However, AsV exposure often represses

358

the stimulatory effects on P transporters to P starvation.49 This down-regulation of expression

359

may be a protecting strategy for plants to reduce AsV uptake. However, PvPht1;3 expression

360

was not affected by AsV under either +P or –P condition (Figure 5C), indicating that PvPht1;3

361

was stable under AsV pressure. Steady-state expression of PvPht1;3 may be critical for AsV

362

uptake and As hyperaccumulation in P. vittata, this way, its efficient AsV and P uptakes are

363

unaffected by AsV.

364

Besides transcriptional expression, tissue specific expression pattern was also crucial for

365

functional analysis of PvPht1;3. We generated transgenic soybean and A. thaliana plants

366

expressing GUS with the PvPht1;3 promoter (Figure 5DE).50 In transgenic soybean plants

367

grown in P-sufficient condition, cross (Figure 5D-i) and longitudinal (Figure 5D-ii) section

368

revealed that the GUS staining was primarily in the stele cells, with no GUS staining being

369

observed at cortex or epidermis. Similarly, in transgenic A. thaliana plants, GUS signal was

370

also mainly in stele when harbored in GUS reaction mix for 2 h (Figure 5E-ii). With longer

371

time, GUS signal was also observed in other root tissues, but much lighter than stele cells

372

(Figure 5E-iii). Gene expression in two different plant species clearly showed that GUS driven

373

by PvPht1;3 promoter was mainly expressed in plant root stele cells, indicating that PvPht1;3

374

was mainly expressed in the root steles in P. vittata.

375

In transgenic tobacco lines with 35S promoter, PvPht1;3 was expressed constitutively in

376

all root cells, indicating AsV translocation was strongly promoted by PvPht1;3 (Figures 3 &

377

4). In P. vittata, PvPht1;3 was likely specifically expressed in the steles (Figure 5), indicating

378

that PvPht1;3 may play a key role in efficient translocation of AsV. However, still unknown

379

are the mechanisms regarding its efficient As transfer from the roots to fronds in P. vittata. Su

380

et al. reported that P. vittata mainly loaded AsIII into the stele as AsIII was the main form in 15



Page 16 of 26

381

the xylem.51 This was also supported by the efficient AsV reduction in the roots/rhizomes of

382

P. vittata.52-54 However, AsV is also transported to the fronds in xylem sap.55 P. vittata often

383

grows in aerobic soils, with AsV as the main form entering the roots. In addition, AsV was

384

concentrated primarily in the roots of P. vittata.51,54,55 Increase of P in growth media inhibits

385

the efficient As transfer to the fronds of P. vittata.22 The results indicated that AsV was also

386

translocated to the stele of P. vittata. In this study, functional identification of PvPht1;3

387

provided new perspectives regarding As translocation in P. vittata. PvPht1;3 had stronger

388

affinity for AsV than other plant P transporters, and its expression in the stele was higher than

389

other root tissues. It was likely that PvPht1;3 participated in AsV loading into the xylem,

390

suggesting that, beside AsIII, P. vittata was efficient to transfer AsV to root xylem sap.

391

Combining strong AsV absorption, efficient As translocation and high As tolerance, P.

392

vittata can be effective in phytoremediation of As-contaminated soils. This study showed that

393

P transporter PvPht1;3 played a crucial role in efficient AsV transport from the roots to fronds.

394

Expressing PvPht1;3 enhanced As acquisition and accumulation in plants, thus has a potential

395

to enhance As accumulation in plants to improve phytoremediation.

396 397

ACKNOWLEDGMENTS

398

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

399

21637002 and 41807118) and the National Key Research and development program of China

400

(Grant No. 2016YFD0800801). We thank Prof. Huixia Shou at Zhejiang University for

401

providing the yeast mutant line and expression vector. We also thank Professor Hong Liao at

402

Fujian Agriculture and Forestry University for providing the Agrobacterium rhizo-genes strain

403

K599.

16


Page 17 of 26

404 405


Table 1. Parameters of AsV uptake kinetics of PvPht1;3 expression tobacco lines and wildtype plants under hydroponic solution. PvPht1;3Ex

Vmax

Km

(nmol g-1 root DW min-1)

(μM)

WT -P

28.9±5.3

Ex1 -P

tobacco line

Linear Slope

r2adj

22.4±8.8

/

0.96

37.7±2.1

13.5±1.9

/

0.99

Ex2 -P

40.8±6.1

25.8±7.9

/

0.98

Ex3 -P

53.3±9.1

29.5 ±9.86

/

0.98

WT +P

/

/

0.077±0.003

1.00

/

/

0.077±0.008

0.98

/

/

0.092±0.013

0.96

/

/

0.078±0.010

0.97

and P treatment

Ex1 +P Ex2 +P Ex3 +P

406 407

17



Page 18 of 26

408 409

Figure 1. Transport activity of PvPht1;3 and PvPht1;2 for P and AsV in yeast. (A) AsV

410

tolerance of yeast cells expressing PvPht1;2 compared to PvPht1;3 under 500 μM AsV; (BC)

411

Growth curves of yeast P transporter mutant strain ΔEY917 transformed with either vector

412

containing PvPht1;2 (B) / PvPht1;3, where PvPht1;2 and PvPht1;3 transformants were grown

413

in medium containing 500 µM AsV (AsV+) or 0 µM AsV (AsV-). (C). Optical density (OD)

414

(Abs600) measurements during logarithmic growth to generate exponential trend lines (y(x) =

415

a*ekx), k means the growth rate coefficient; (D) Michaelis constants (Km) of AsV for yeast

416

mutant strain ΔEY917 expressing PvPht1;2 or PvPht1;3; and (E) Arsenic accumulation of

417

yeast cells expressing PvPht1;2 or PvPht1;3, where PvPht1;2 and PvPht1;3 transformants were

418

grown for 3 h in medium with 1.5 mM P + 150 µM AsV. Values are the mean SD of three

419

independent assays.

420 18


Page 19 of 26


421 422

Figure 2. P and arsenic accumulation in PvPht1;3-expressing lines and wild-type (WT)

423

tobaccos. (A) Relative expression of PvPht1;3 in transgenic lines and WT tobaccos by

424

quantitative real time PCR (qRT PCR); (B) P concentrations in the roots and shoots of

425

transgenic lines and WT tobaccos; (C, D) Arsenic concentrations in the roots (C) and shoots

426

(D) of transgenic lines and WT tobaccos in short-term hydroponic experiments, As

427

concentrations were analyzed in tobacco seedlings exposed to 20 μM AsV for 1 d; (E) Arsenic

428

translocation factor of PvPht1;3 lines compared to WT tobaccos. Transgenic plants and WT

429

plants were exposed to 20 μM AsV for 1 d. Values are means ± SE (n = 4) and asterisk indicates

430

that the values for PvPht1;3 lines differ significantly (*P < 0.05;) compared with WT plants.

431

FW, fresh weight, and DW, dry weight.

432

19



Page 20 of 26

433 434 435 436

Figure 3. (A) Uptake kinetics of AsV in PvPht1;3 line Ex1 and wild-type (WT) tobaccos under

437

+P and -P regimes (100 µM). Values are means ± SE (n = 4). DW, dry weight. (B) Arsenic

438

speciation in the xylem sap of PvPht1;3 lines and WT tobaccos. After exposing to 20 μM AsV

439

for 1 d, the xylem sap of tobacco seedlings was collected by pipet from the cut surface for 2 h.

440

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

441

differences (p < 0.05).

20


Page 21 of 26


442 443

Figure 4. Biomass and arsenic accumulation in the PvPht1;3 lines and WT plants cultivated in

444

As-contaminated soil. The 2-week stage PvPht1;3 lines and WT tobacco seedlings were grown

445

in As-contaminated soil containing 53 mg kg-1 As for 30 d. Values are means ± SE (n = 6) and

446

asterisk indicates that the values for PvPht1;3 lines differ significantly (*P < 0.05) compared

447

with WT plants. DW, dry weight.

448

21



Page 22 of 26

449 450

Figure 5. Expression pattern and tissue localization of PvPht1;3. (A,B) Transcriptional patterns

451

of PvPht1;3 in the roots and fronds of P. vittata seedling under 0.2X Hoagland nutrient solution

452

(HNS) by semi-quantitative RT PCR (A), or qRT-PCR (B); (C) Transcriptional levels of

453

PvPht1;3 in the roots responding to P-deficiency or AsV exposure. P. vittata sporophytes were

454

exposed to 0.2X HNS containing 100 μM KH2PO4 (P+As−), 0 μM KH2PO4 (P−As−), 0 μM

455

KH2PO4/50 μM Na2HAsO4·7H2O (P-As+) ,or 100 μM KH2PO4/50 μM Na2HAsO4·7H2O

456

(P+As+) for 3 d. Housekeeping gene, PvActin, was used as an internal standard. Error bars

457

indicate SE of three biological replicates; (D) GUS staining observation of transgenic soybean

458

roots harboring PvPht1;3 promoter::GUS fusion. Hand-cut cross section (i) and whole tissue

459

(ii) of primary root of transgenic soybean was assayed under 0.2X HNS solution. Bar = 2 mm;

460

(E) GUS staining observation of transgenic A. thaliana harboring PvPht1;3 promoter::GUS

461

fusion. (i) Whole seedling of transgenic A. thaliana was assayed under 0.2X HNS solution. (ii

462

and iii) Primary root elongation zone was observed at 0.5h (ii) and 2h (iii) after the reaction.

463

Pink triangles indicate stele cells. Bar = 5 mm in (i), = 0.5mm in (ii and iii).

464

22


Page 23 of 26

465 466 467


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