Identification and characterization of silicon efflux transporters in horsetail (Equisetum arvense)

Journal of Plant Physiology 200 (2016) 82–89

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Identification and characterization of silicon efflux transporters in horsetail (Equisetum arvense) Julien Vivancos a , Rupesh Deshmukh a , Caroline Grégoire a , Wilfried Rémus-Borel a,1 , Franc¸ois Belzile b , Richard R. Bélanger a,∗ a Département de Phytologie–Faculté des Sciences de l’agriculture et de l’alimentation, Centre de recherche en horticulture, Université Laval, Pavillon Paul-Comtois, Québec, G1V 0A6 QC, Canada b Département de Phytologie–Faculté des Sciences de l’agriculture et de l’alimentation, Université Laval, Pavillon Charles-Eugène-Marchand, Québec, G1V 0A6 QC, Canada

a r t i c l e

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Article history: Received 4 May 2016 Received in revised form 16 June 2016 Accepted 16 June 2016 Available online 18 June 2016 Keywords: Silicon transport Equisetum arvense Lsi2 genes Triticum aestivum Efflux transporter Xenopus oocytes

a b s t r a c t Silicon (Si) is a beneficial element to plants, and its absorption via transporters leads to protective effects against biotic and abiotic stresses. In higher plants, two groups of root transporters for Si have been identified: influx transporters (Lsi1) and efflux transporters (Lsi2). Lsi1 transporters belong to the NIPIII aquaporins, and functional Lsi1s have been found in many plants species. Much less is known about Lsi2s that have been characterized in only a few species. Horsetail (Equisetum arvense), known among the highest Si accumulators in the plant kingdom, is a valuable model to study Si absorption and deposition. In this study, we first analyzed discrete Si deposition patterns in horsetail shoots, where ubiquitous silicification differs markedly from that of higher plants. Then, using the sequenced horsetail root transcriptome, two putative Si efflux transporter genes, EaLsi2-1 and EaLsi2-2, were identified. These genes share low sequence similarity with their homologues in higher plants. Further characterisation of EaLsi21 in transient expression assay using Nicotiana benthamiana epidermal cells confirmed transmembrane localization. In order to determine their functionality, the EaLsi2-1 was expressed in Xenopus oocytes, confirming that the translated protein was efficient for Si efflux. Both genes were equally expressed in roots and shoots, but interestingly, showed a much higher expression in the shoots than in the roots in contrast to Lsi2s found in other plants, a result consistent with the specific anatomy of horsetail and its rank as one of the highest Si accumulators among plant species. © 2016 Elsevier GmbH. All rights reserved.

1. Introduction Silicon (Si) is ubiquitous in nature as the second most abundant element in the soil. Silicon is absorbed by plants in its soluble form, silicic acid, yielding remarkable beneficial effects (Epstein, 1994). The absorption and accumulation of Si protect plants against several biotic and abiotic stresses, including fungal diseases, insects,

Abbreviations: Si, silicon; PCR, polymerase chain reaction; NCBI, national center for biotechnology information; RT-PCR, reverse transcriptase PCR; CDS, coding DNA sequence; cDNA, complementary DNA; cRNA, complementary RNA; GFP, green fluorescent protein; DEPC, diethylpyrocarbonate; RACE, rapid amplification of cDNA ends. ∗ Corresponding author at: Département de Phytologie, pavillon Paul-Comtois, local 3305 2425 rue de l’Agriculture, Université Laval, Québec, G1V 0A6 QC, Canada. E-mail address: [email protected] (R.R. Bélanger). 1 Jade International, Cadéra South Park, Avenue Ariane – CS 60027, 33693 Merignac, France. http://dx.doi.org/10.1016/j.jplph.2016.06.011 0176-1617/© 2016 Elsevier GmbH. All rights reserved.

drought, lodging, salinity and nutrient imbalance (Epstein, 1999; Fauteux et al., 2005; Liang, 1999; Liang et al., 2015; Rémus-Borel et al. 2005). Despite its beneficial effects, Si is not considered as an essential element for plants, since it is dispensable for the completion of the life cycle of most species, with the significant exception of horsetail (Chen and Lewin, 1969). In plants, the final concentration of this element varies between 0.1 to 10% Si in top dry weight, with the strongest Si accumulators belonging to the orders Poales and Equisetales (Hodson et al., 2005). Most importantly, the plant species that accumulate the highest amounts of Si seem to benefit the most from its protective effects (Epstein, 1999; ArsenaultLabrecque et al., 2011). Plants take up Si from the soil solution as silicic acid [Si(OH)4 ], through transporters recently discovered in rice (Ma et al., 2006). The Si transport from the soil solution to the shoots involves influx and efflux by two different families of proteins (Ma, 2010). Firstly, Si influx transporters, also known as Lsi1, are passive channels belonging to the NIP subfamily (nodulin 26-like proteins)

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of aquaporins (Gomes et al., 2009; Deshmukh and Bélanger, 2015). These transporters allow the passive passage of silicic acid from the soil solution to the root cells. Lsi1 homologues have been identified originally in rice (Oryza sativa; Ma et al., 2006) and in many other plant species since (Chiba et al., 2009; Mitani et al., 2009a; Mitani et al., 2011; Deshmukh et al., 2013; Deshmukh et al., 2015). Functional Lsi1s have been reported to be essential for a plant species to accumulate Si (Deshmukh et al., 2015). Secondly, Si efflux transporters, also called Lsi2, are transmembrane proteins with 9–12 transmembrane domains, and belong to the less-studied family of putative anion transporters. Lsi2s are believed to be active transporters, driven by the proton gradient, based on experiments using protonophores (Ma, 2010). Unlike Lsi1s, only a few Si efflux transporters have been discovered so far in higher plants, namely in rice, barley, maize and pumpkin (Ma et al., 2007; Mitani et al., 2009b; Mitani-Ueno et al., 2011). Si transporters were also discovered in the salt-water unicellular diatoms, (Hildebrand et al., 1997) but have no sequence similarity with those from plants. Equisetum is generally considered a living fossil and has great importance in ecological and evolutionary studies. The genus Equisetum is monophyletic, with around 30 species, and is a pteridophyte, a relative of living ferns (Des Marais et al., 2003). Equisetum arvense (horsetail) has been used as a model to study silicon mineralization in land plants because of its extremely high Si content (Bauer et al., 2011). But until recently Si transport mechanism in horsetails was totally unknown at the molecular level probably due to the lack of resources and a very large genome of 14 Gb (Bainard et al., 2011; Grégoire et al., 2012). We recently identified a multigene family of horsetail Si influx transporters that have distinct characteristics from those of higher plants (Grégoire et al., 2012). In this study, we report the discovery of two unique Si efflux transporters from horsetail. The properties and Si efflux activity of these proteins were investigated using plant and Xenopus oocytes heterologous expression systems. Our results support the concept that these Si efflux transporters, in conjunction with the numerous Si influx transporters, explain the outstanding Si accumulation of horsetail. 2. Materials and methods 2.1. Plant material Horsetail material was collected from a natural colony of asexually reproducing plants growing at the Jardin Van den Hende botanical garden on the Université Laval campus, Québec, Canada. For RNA and genomic DNA extractions, roots were collected from a single plant in late June, during a period of active growth. For the Si uptake experiment (see below), horsetail, Arabidopsis and rice plants were grown in a greenhouse under a 16 h/8 h photoperiod, 22 ◦ C day/18 ◦ C night, 80% humidity, in 20 cm pots containing commercial potting mix (Connaisseur Premium Potting Soil, Fafard, http://www.fafard.com) and fertilized twice a week with a modified Hoagland solution containing 1.7 mM Si as potassium silicate (Kasil #6, National Silicates, http://www.silicates. com; Guével et al., 2007). Potassium levels in the Hoagland solution were adjusted in control conditions to compensate for the additional input of K from potassium silicate. 2.2. Scanning electron microscopy (SEM) and X-ray microanalysis mapping Scanning electron microscopy and X-ray microanalysis mapping were used to locate Si deposition in soil-grown horsetail fed with Si for 30 days. Shoot samples were prepared as described by Guével et al. (2007). Briefly, shoots were lyophilized and coated with gold

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and palladium to provide conductivity to the samples. Samples were analyzed using a CAMECA SX-100 Universal EPMA microscope (Cameca instruments Inc, www.cameca.com) operating at a voltage of 15 kV and a current of 20 nA.

2.3. Nucleic acid extraction and cDNA synthesis Horsetail total RNA was extracted from roots and shoots using TRIzol reagent (Invitrogen, http://www.invitrogen.com) according to the manufacturer’s instructions, checked for integrity on a denaturing agarose gel, and stored at −80 ◦ C until use. Arabidopsis total RNA was extracted from roots and shoots using an RNA purification kit (Qiagen, http://www.qiagen.com) and stored at −80 ◦ C until use. Horsetail genomic DNA were extracted from leaves and stems using a DNeasy plant mini kit (Qiagen), and stored at −80 ◦ C until use. For horsetail, Arabidopsis and rice, first-strand cDNAs were prepared from 1 ␮g total RNA treated with RQ1 RNase-free DNase (Promega, http://ww.promega.com), then reverse-transcribed using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT)18 primers.

2.4. Identification and cloning of horsetail EaLsi2 genes A horsetail transcriptome database was constructed as described in Grégoire et al. (2012). To identify possible homologues of Lsi2 in horsetail, the assembled contigs were searched by tBLASTn using known full-length Lsi2 sequences from rice (Ma et al., 2006) and pumpkin (Mitani-Ueno et al., 2011). The candidate EaLsi2-1 and EaLsi2-2 full-length coding sequences (CDS) were amplified using the high-fidelity Phusion DNA polymerase (New England Biolabs, http://www.neb.com) from horsetail root cDNA using specific primers listed in Supplementary Table 1. Full length CDS were amplified and subcloned in the pGEM-T-Easy vector (Promega, www.promega.com). Subsequent Sanger sequencing of cloned products on a 3730xl DNA Analyzer (Applied Biosystems, http://www.appliedbiosystems.com) revealed two distinct CDS. These constructions were named pGEM.EaLsi2-1 and pGEM.EaLsi22. For each CDS, eight clones were sequenced. To obtain full-length cDNA sequences, both 5 and 3 RACE (Frohman et al., 1988) were performed using HotMaster Taq DNA (5 PRIME, http:// www.5prime.com). The amplification products were subcloned into pGEM-T Easy vector. For each construction, ten clones were sequenced. After sequencing, RNA-Seq reads were mapped back to the cloned cDNA sequences using CLC Genomic Workbench (http:// www.clcbio.com/) to correct any sequencing or enzymatic errors. This also allowed a digital comparison of expression levels of both Lsi2 genes in roots. To sequence the full-length EaLsi2 genes, PCR was performed on genomic DNA using Phusion DNA polymerase and the same primers used for CDS cloning (Supplementary Table 1). A 7 kb PCR product was visualized on an agarose gel, and subcloned into pGEM-T Easy vector. For each gene, 12–20 clones were sequenced using internal primers that were designed as needed. To identify the intron–exon gene structure, full gene sequences were compared using Spidey (http://www.ncbi.nlm.nih.gov/spidey) for perfect sequence match with the corresponding cloned cDNA. The translated EaLsi2-1 protein sequence was used to perform a BLASTp search against the nr database at the National Center for Biotechnology Information (NCBI; http://blast.ncbi.nlm.nih.gov/Blast.cgi). Reverse-transcription (RT-PCR) was performed on horsetail root and shoot cDNAs using primers EaLsi2 F/EaLsi2 R535 (Supplementary Table 1). The resulting PCR products were separated on a 2% agarose gel. Since these primers targeted both genes, the resulting product from shoots was digested with the restriction enzyme TaqI to distinguish between the two sequences.

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2.5. Alignments, phylogenetic tree and pairwise comparisons All amino acid alignments were performed with Clustal X (http://www.clustal.org). The transmembrane domains were predicted using the TMHMM server (http://www.cbs.dtu.dk/services/ TMHMM). Signal peptides were predicted with SignalP (http:// www.cbs.dtu.dk/services/SignalP/) and subcellular localization was predicted with TargetP (http://www.cbs.dtu.dk/services/ TargetP/). Protein homology based three-dimensional (3D) structure of EaLsi2-1 generated by using I-TASSER server (http:// zhanglab.ccmb.med.umich.edu), and docking was performed using PatchDock server (http://bioinfo3d.cs.tau.ac.il/). The phylogenetic tree was constructed using MEGA5 version 5.05 (Tamura et al., 2011) using the bootstrap maximum likelihood method with 1000 replicates. Horsetail and higher plant Lsi2 protein sequences were subjected to pairwise comparisons after alignment. Identity and similarity percentages are shown in Supplementary Table 2. 2.6. Sub-cellular localization of EaLsi2-1 transporters in Nicotiana benthamiana leaves The sub-cellular localization of EaLsi2-1 was assessed with the following construct. The EaLsi2 coding region (excluding the stop codon) was amplified using gene-specific primers (Xb-EaLsi2 and Bg-EaLsi2; see Supplementary Table 1) from horsetail root cDNA. The plasmid pGEM.EaLsi2-1 was digested with NcoI and SpeI to release the EaLsi2-1 CDS. This fragment was then inserted in the corresponding site in pCAMBIA1302 (CAMBIA, http://www.cambia. org) generating an in-frame fusion with the GFP coding region, regulated by the CaMV35S promoter. This construct was named 35S:EaLsi2-1:GFP. As a control, pCAMBIA1302 carrying only the 35S promoter and the GFP CDS was used. These two constructs were transformed into the GV3101/pMP90 Agrobacterium tumefaciens strain. Transient expression in Nicotiana benthamiana leaves was performed as described by Johansen and Carrington (2001). Agroinfiltration was performed including another A. tumefaciens strain containing the pGD-p19 plasmid, which expresses the Tomato Bushy Stunt Virus (TBSV) p19 coding region to minimize host RNA silencing (Bragg and Jackson 2004). A. tumefaciens cultures containing 35S:GFP or 35S:EaLsi2-1:GFP were mixed in equal proportions with the A. tumefaciens pGD-p19 culture and co-infiltrated on the abaxial surface of the leaves. Leaves were harvested five days following agroinfiltration and observed by fluorescence microscopy (Olympus BH2 RFCA, http://microscope.olympus-global.com).

Ultra kit (Ambion, http://www.invitrogen.com/site/us/en/home/ brands/ambion.html). Complementary RNAs (cRNAs) were purified using phenol/chloroform precipitation, and suspended in water treated with 0.1% DEPC (Sigma-Aldrich, http://www.sigmaaldrich. com/). Defolliculated stage V–VI oocytes were injected with 25 nl of a 1.0 mM Si solution (control), or with 25 nl of 500 ng/␮l cRNAs solubilized in a 1.0 mM Si final solution in form of potassium silicate. A first pool of 10 oocytes for each treatment of injection was recovered (=T0), rinsed in sucrose-HEPES solution and frozen until Si intracellular measurement. Remaining eggs were maintained at 18 ◦ C in modified Barth medium (MBS) (88 mM NaCl, 1 mM KCI, 2.4 mM NaHCO3, 0.82 mM MgSO4 , 0.33 mM Ca(N03 )2 ·4H2 0, 0.41 mM CaC12, 15 mM Hepes, pH 7.6) supplemented with 100 ␮M of penicillin/streptomycin. Seventy-two hours after injection, a second pool of 10 oocytes for each treatment were recovered, rinsed in sucrose-HEPES solution and frozen until Si intracellular measurement. 2.9. Dosage of Si in Xenopus oocytes Concentrated nitric acid (25 ␮l) was added to each pool of 10 oocytes, which were then dried for 2 h at 82 ◦ C. Plasma-grade water (100 ␮l) was added, and samples were incubated for 1 h at room temperature. Samples were vortexed, then centrifuged for 5 min at 13,000g. The intracellular Si concentration was measured in 10 ␮l of supernatant by Zeeman atomic absorption using a Zeeman atomic spectrometer AA240Z (Varian; http://www.varian. com) equipped with a GTA120 Zeeman graphite tube atomizer. The standard curve was obtained using a 1000 ppm ammonium hexafluorosilicate solution (Fisher Scientific, http://www.fishersci. com). Data were analyzed with SpectrA software (Varian). 3. Results 3.1. Si deposition pattern in horsetail shoots X-ray microanalysis of horsetail grown in soil and supplemented with Si revealed a distinct pattern of Si deposition. The scanning electron microscopy image of shoots (Fig. 1B, left panel) showed a double offset row of stomata, and light-colored raised pilulae, where Si is more densely deposited. In Fig. 1B (right panel), a color scale of Si deposition was used, with blue indicating low Si and red high Si deposition. Black areas, such as the stomatal aperture, indicated no Si deposition.

2.7. Plasmid constructions for heterologous expression in Xenopus oocytes

3.2. Identification and cloning of EaLsi2-1 and EaLsi2-2 efflux Si transporters

Fragments containing OsLsi2 and EaLsi2-1 CDS were excised from pGEM.OsLsi2 and pGEM. EaLsi2-1 by EcoRI/XbaI digestion and inserted into EcoRI/XbaI pre-digested Pol1 vector, a Xenopus laevis oocyte expression vector derived from pGEM and comprising the T7 promoter, the Xenopus globin untranslated regions and a poly(A) tract (Caron et al., 2000). All vectors were transformed into Escherichia coli TOP10 strain and stored at −80 ◦ C. Integrity of the constructs was checked by sequencing prior to in vitro translation.

We first attempted to identify horsetail Si efflux transporters by homology cloning using Lsi2 sequences from higher plants, but this approach yielded no results. We then successfully used a root RNA-Seq data to identify the transporters described in this study. After a first round of BLAST searches using known higher plant Lsi2 sequences, two horsetail contigs appearing to code for complete CDS were identified in the root transcriptome along with several short, and partial sequences. A second iterative round of BLASTn was performed with the two long horsetail contigs, but did not allow identification of any new related sequences. These two sequences were used as a starting point to design PCR primers (Supplementary Table 1). PCR was performed on the same root cDNA used for transcriptome sequencing. Two 1593-bp products were amplified with a high-fidelity polymerase and sequenced. None were identical to the in silico contigs, indicating that these assemblies might be chimeric. Full-length cDNA sequences were obtained by RACE-PCR, yielding longer sequences than the in silico contigs, possibly because assembly of UTRs was incomplete.

2.8. Si transport assays using heterologous expression in Xenopus oocytes Plasmids containing either the OsLsi2 or EaLsi2-1 CDS were recovered from a fresh bacterial culture using a QIAprep Spin Miniprep kit (Qiagen). 5 ␮g of each plasmid were linearized using NheI (Roche, http://www.roche.com). Digested products were column-purified using a PCR purification kit (Qiagen), and 1 ␮g of DNA was transcribed in vitro using the mMessage mMachine T7

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Fig. 1. Horsetail growth habit and Si deposition pattern. (A) Horsetail plant from a natural colony (B) SEM and X-ray microanalysis mapping images showing Si deposition on horsetail shoots following feeding with potassium silicate. The concentration of Si is indicated by color, where red represents the highest concentration of Si and black indicates no Si. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

However, 5 RACE of EaLsi2-1 was unsuccessful, possibly due to secondary structure in the 5 end of the mRNA. Therefore, the 5 UTR sequence for this gene comes only from the in silico contig. To confirm sequences, RNA-Seq reads were mapped back to both horsetail cDNAs (Fig. 2A). Digital expression levels in horsetail roots were similar for both cDNAs, with a mean read coverage of 56 for EaLsi21 and 82 reads for EaLsi2-2. After sequencing, the EaLsi2-1 gene was found to be 7.3 kb long. While the exon sequences matched the cloned cDNA, SNPs and minor insertions/deletions were found only in the two introns, indicating the possible presence of allelic variants of the same gene. One of the full-length alleles of EaLsi2-1 is shown in Fig. 2B. Using RT-PCR and conserved primers, it was shown that both EaLsi2-1 and EaLsi2-2 are expressed in horsetail roots and shoots (Fig. 2C). Digesting the shoot PCR product with TaqI allowed to distinguish the two transcripts and revealed that both are present in the shoots. Both horsetail Lsi2 proteins were predicted to have nine transmembrane domains (Fig. 3A, Supplementary Fig. 1). Blast search performed at Protein Data Bank (PDB) revealed that there is no known protein with available

three-dimensional (3D) structure having sequence similarity with horsetail Lsi2 (http://www.rcsb.org/pdb/home/home.do). Therefore, 3D structure of EaLsi2-1 generated by using the iterative threading assembly refinement (I-TASSER) server (http://zhanglab. ccmb.med.umich.edu), and subsequent docking performed using PatchDock server (http://bioinfo3d.cs.tau.ac.il/) showed binding site for Si (Supplementary Fig. 2). A BLASTp search using EaLsi21 found that the closest sequence is from Arabidopsis: the divalent ion transporter At1g02260 (Fig. 3B).

3.3. Horsetail Lsi2s are distinct from higher plant Si efflux transporters The two horsetail Lsi2 CDS have similar sequences that differ at only 43 positions (SNPs) out of 1593 bp. Most of these mutations are silent however, as the proteins have only 13 different amino acids out of 530, which makes them 97% identical (Fig. 3A). However, horsetail Lsi2s share only 45–49% identity with cereal and

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Fig. 2. Gene structure, cDNA coverage and expression pattern of EaLsi2 genes. (A) Gene structure of one fully sequenced allele of EaLsi2-1. (B) Read coverage of EaLsi2-1 and EaLsi2-2 cDNAs in roots. (C) Expression pattern of EaLsi2-1 and EaLsi2-2 in horsetail roots and shoots grown in absence (Si-) or presence (Si+) of silicon; shoot product digested with TaqI. Based on Student’s t-test (p < 0.05), there is no significant difference in expression levels between the genes or Si conditions in either roots or shoots.

pumpkin proteins (Supplementary Table 2), which separates them in the phylogenetic tree (Fig. 3B, Supplementary Fig. 3). 3.4. EaLsi2-1 localizes at the plasma membrane Transient expression of EaLsi2-1 fused with GFP was performed in N. benthamiana epidermal cells to identify its subcellular localization. When expressed alone, the GFP protein showed fluorescence in the nucleus, cytosol and plasma membrane (Fig. 4A), while the EaLsi2-1-GFP fusion protein was observed only at the plasma membrane (Fig. 4B). 3.5. Si efflux activity in Xenopus oocytes We tested the Si transport activity of one of the two horsetail Lsi2s along with the rice Lsi2 as a positive control. After 72 h of incubation, both proteins allowed a significant efflux of Si (14% Si efflux for rice, and 19% Si efflux for horsetail) (Fig. 5). No Si efflux was detected in control oocytes injected only with Si, but not with a transporter, confirming that there is no intrinsic Si efflux in untransformed oocytes. 4. Discussion The element Si is present in abundant amounts in nature, soils and oceans. Particularly in plants, several reports link Si supply with greater resistance to abiotic and biotic stresses (Epstein, 1999; Fauteux et al., 2005). Still, many questions remain in respect to its absorption by living organisms. Horsetail, one of the highest Si accumulating plants (Hodson et al., 2005), is a valuable model to unravel the role of transporters in Si biology. Yet, very large genome and overall lack of resources for horsetail pose certain experimental challenges. In this paper, we have identified two unique Si efflux transporters via the sequencing of the horsetail root transcriptome. These transporters appear to have unique properties that would contribute to horsetail’s ability to accumulate large concentrations of Si. The deposition pattern of Si in horsetail shoots is strikingly different from the pattern earlier observed in higher plants. Cereals in particular show high Si deposition in discrete locations: namely specialized cells called silicaphile cells, at the base of trichomes, and in guard cells (eg. wheat; Côté-Beaulieu et al., 2009). In contrast,

horsetail shows abundant and widespread Si deposition, particularly at the raised epidermal pilulae (Fig. 1B right panel, visible in red color), while subsidiary cells of stomata (in blue) have lower Si deposition. Our results are in accordance with earlier reports that show a stronger deposition of Si in these pilulae (Currie, 2007). The double offset rows of stomata are characteristic of horsetail (Perry and Fraser, 1991). EaLsi2 proteins were predicted to have nine transmembrane domains. Localization in the plasma membrane was later confirmed experimentally for EaLsi2-1 by transient expression in N. benthamiana epidermal cells. This is consistent with all other Lsi2 proteins, which are all reported to be transmembrane proteins. Some similar higher plant efflux transporters, such as the boron efflux transporter BOR1 (Takano et al., 2005), show a polar localization in the specialized cells of the Casparian strip, but they show apolar localization when expressed in N. benthamiana leaf cells, because these cells lack a Casparian strip allowing proper subcellular localization. Therefore, it is not possible to infer the in planta subcellular localization from transient expression data. The Lsi2 proteins belong to a family of putative anion transporters, but Si is absorbed by plants in an uncharged, non-anionic form (Broadley et al., 2011). Interestingly, silicic acid and boric acid are the two molecules absorbed by plants in an uncharged form (Miwa and Fujiwara, 2010) and both are transported inside roots using a similar combination of polar-localized influx and efflux transporters (Miwa et al., 2009; Zangi and Filella, 2012). Passive influx transporters are similar for boric acid and silicic acid (NIP aquaporins) while proton gradient driven BOR1 and Lsi2 do not have related sequences (Takano et al., 2005; Ma et al., 2007). The two elements also differ in overall accumulation: no Si toxicity has been reported even at high concentrations (Epstein, 1999) while boron absorption has to be tightly regulated (Miwa and Fujiwara, 2010). Horsetail Lsi2s have several characteristics that set them apart from higher plant silicon efflux transporters. First, the sequences share less than 50% identity at the amino acid level. However, it has 51% identity with its closest homologue At102260 from Arabidopsis, which was investigated for its arsenite transport capacity, but not for Si, when expressed in yeast (Bienert et al., 2008). The structure of the horsetail gene is also different from all the previously described Lsi2 genes, which have a single intron, while the horsetail Lsi2s have two introns. Both horsetail genes were equally

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Fig. 3. Alignment and phylogenetic relationship of Lsi2 proteins. (A) Alignment of two horsetail Lsi2 proteins. The nine putative transmembrane domains are indicated by gray boxes lines above the sequences. Signal peptides (positions 1–31) are in lowercase. (B) Phylogenetic tree of known Lsi2 proteins from horsetail and higher plants. The species acronym is included before the gene name: At, Arabidopsis thaliana; Cm, Cucurbita moschata; Ea, Equisetum arvense; Hv, Hordeum vulgare; Os, Oryza sativa; Zm, Zea mays.

expressed in roots and shoots suggesting that they both contributed to Si transport. Of particular interest, they showed a much higher level of expression in the shoots than in the roots, in clear contrast from previous reports that showed similar or lower expression of Lsi2s in the shoots (Ma et al., 2007; Mitani-Ueno et al., 2011; Yamaji et al., 2012). Similarly, horsetail Lsi1 (EaNIP3;1) has relatively higher expression in the shoot (Grégoire et al., 2012) while Lsi1 expression in rice and barley was found to be restricted to the roots (Ma et al., 2006; Chiba et al., 2009). These differences could very well explain the higher Si absorption of horsetails in leaf/aerial tissues. This distinct feature is likely linked to the specific anatomy of horsetail, which has characteristic jointed, ridged stems, microphyllous leaves, and root whorls at rhizome nodes; large carinal canals are also thought to be water-conducting channels (Leroux et al., 2011), which would carry Si to the nodes and aerial parts of the plant.

Given the absence of a transformation system for horsetail, and our inability to obtain distinctive phenotypes when transforming Arabidposis with EaLsi2 (results not shown), it was a challenge to assess functionality of a gene in horsetail. This is why the oocyte transport assay was a practical option to determine if EaLsi2-1 was indeed capable of Si efflux. This heterologous expression system is well suited for the study of membrane transport proteins (Haferkamp and Linka, 2012) and has proven reliable in most studies trying to assess functionality of Si transporters (Ma et al., 2006, 2007; Grégoire et al., 2012; Mitani et al., 2011; Deshmukh et al., 2015; Montpetit et al., 2012). Using rice Lsi2 as a positive control, our results confirmed that EaLsi2 was a functional efflux transporter of Si. Previous studies have shown that the maize, barley and pumpkin Lsi2s had an efflux activity of 1.6%, 15% and 15% respectively (Mitani et al., 2009b; Mitani-Ueno et al., 2011) but different

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Fig. 4. Transient expression of EaLsi2-1 in Nicotiana benthamiana cells showing localization in the plasma membrane. (A) GFP alone or (B) fused to the C-terminus end of EaLsi2-1 (EaLsi2-1-GFP) were expressed in Nicotiana benthamiana leaves and observed by fluorescence microscopy. Scale bars represent 50 ␮m.

Acknowledgements The project was funded by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) in collaboration with Syngenta Biotechnology and the Canada Research Chairs Program to R.R.B., C.G. was supported by a Canada Graduate Scholarship from NSERC and a Doctoral Research Scholarship from the Fonds de Recherche Nature et Technologie du Québec. The authors would like to thank Dr. P. Isenring’s Nephrology Research Group of the Centre Hospitalier Universitaire de Québec/L’Hôtel-Dieu de Québec Institution for help with the oocyte assays.

Appendix A. Supplementary data Fig. 5. Efflux activity of horsetail Si transporter in Xenopus oocytes. Si efflux was measured in Xenopus oocytes injected either with only Si (control) or Si and cRNA for horsetail transporter EaLsi2-1. Before sampling, oocytes were incubated for 0 or 72 h in Barth medium without any added Si. Values are means ± standard error (n = 3).

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph.2016.06. 011.

References conditions, namely the use of 68 Ge as a surrogate for Si in other experiments, prevent from drawing conclusions about the relative activity of the different Lsi2s. For instance, Garneau et al. (2015) compared transport activity of Si and 68 Ge by mammalian aquaporins and found differences that led them to conclude that there was a different affinity for the two substrates. Therefore, the use of Si instead of proxy 68 Ge seems to be more reliable. In conclusion, we have discovered and characterized two Si efflux transporters from horsetail, which share less than 50% identity with their higher plant homologues. Transient expression in N. benthamiana confirmed the transmembrane nature of these little studied putative anion transporters, while heterologous expression in oocytes confirmed the Si efflux function. Both genes were equally expressed in roots and shoots, but interestingly, showed a much higher expression in the shoots than in the roots in contrast to Lsi2s found in other plants, a result consistent with the specific anatomy of horsetail and its rank as one of the highest Si accumulators among plant species.

Conflict of interest The authors declare that they have no conflict of interest.

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