Introduction

Arsenic (As) is a metalloid naturally occurring in the earth’s crust and released in the environment as consequence of erosion, volcanic emissions, etc. Soils can be highly contaminated by As coming mainly from mining and smelting activities, coal combustion and the use of As-containing agrochemicals1. Then As can spread in soil and diffuse in groundwater, entering the food chain through drinking water and contaminated vegetables2. This metalloid is highly toxic to most biological systems and it has been recognized as carcinogenic for human by IARC (International Agency for Research on Cancer)3.

Plant sensitivity to As varies according to species and a relatively low number of plant species are naturally tolerant to As; among them, Pteris vittata and other members of the Pteridaceae are able to hyperaccumulate As without showing any symptom4,5,6,7.

P. vittata L. (Chinese brake fern) is the first discovered As-hyperaccumulating plant4. This fern is able to remove large amounts of As from soil and shows interesting growth characteristics, including large biomass, extensive root system, high growth rate and perennial habit. P. vittata mostly concentrates As in fronds which is a typical feature of hyperaccumulators4,8 even though As concentration in roots can reach 100 mg kg−1 9. Such As concentration can be considered very toxic for the majority of plants, but not for P. vittata which can tolerate up to 10,000 mg kg−1 10.

In the environment, As can exist as inorganic or organic species, but it occurs predominantly in inorganic forms. The arsenate (AsV), the highest oxidized form and the arsenite (AsIII), the highest reduced form, are predominant in aerobic and anaerobic environments, respectively2. The organic species of AsV that are found at low concentrations in most soils include monomethylarsonicacid, dimethylarsinicacid and trimethylarsineoxide2.

In plants exposed to As, AsV is readily reduced, both enzymatically and non- enzymatically, to AsIII. AsV can be directly reduced to AsIII by arsenate reductase (ACR), an enzyme first isolated from bacteria and yeasts11. Despite their common function, the arsenate reductases identified up to now are members of several independent families that have convergently evolved in prokaryotes and eukaryotes. The Saccharomyces cerevisiae arsenate reductase ACR2p12,13,14 belongs to the enzyme family that uses glutaredoxin (GRX) as hydrogen donor. It is homologous to the CDC25A cell-cycle protein phosphotyrosine phosphatase (PTPase)15 and rhodanese, a thiosulphate transferase16. Using functional complementation by phenotypic suppression screening of a P. vittata cDNA library in yeast, a PvACR2 arsenate reductase gene was identified in the fern P. vittata5. Hereafter ACR activity have been detected in the recombinant proteins of Arabidopsis thaliana (AtAsr/AtACR2)17, P. vittata (PvACR2)5 and Oryza sativa (OsACR2.1 and OsACR2.2)18. Very recently a novel arsenate reductase gene HAC1/ATQ1 has been identified in A. thaliana by genome-wide association mapping for the identification of quantitative trait loci related with arsenic accumulation and tolerance19,20. AtACR2 shows phosphatase activity, while the PvACR2 enzyme, like ScAcr2 protein, does not5,18,21. Also like ScAcr2p, the PvACR2 enzyme uses glutathione (GSH) and GRX as electron sources5,18, suggesting that the catalytic cycle involves the formation of a mixed disulfide between GSH and ACR2 that is resolved by GRX11. As a result of this activity, which is considered as the first step in the major As detoxification pathways found in plants22,23, more than 90% of the As in the root and in the shoot is turned into arsenite (AsIII)22,24,25,26.

Moreover, AsV is an analogue of inorganic phosphate (Pi) and is easily transported across the plasmalemma by Pi transporters (PHT)27. This competition between AsV and Pi for the same transport systems has been observed in As hyperaccumulators28,29, As-tolerant non-hyperaccumulators30 and As-sensitive non-accumulators31,32.

Although the presence and the expression level of one arsenate reductase gene (PvACR2) has been characterized in gametophytes5, till now no arsenate reductase protein has been directly observed in P. vittata.

The aim of this work was to characterize two arsenate reductase proteins, PvACR2 and Pv2.5–8 (gi: 89243488) in both gametophytes and sporophytes of P. vittata in order to improve the current knowledge about the metabolic pathways of As detoxification occurring in this plant.

Results

In order to evaluate the impact of a chronic exposure to arsenic, ferns were treated, once a week, for 60 days with 334 μM As, which is a non-lethal dose for P. vittata. Arsenic did not affect frond development. On the contrary, root dry biomass was reduced (−42%) in As-treated plants (Supplementary Table S1, online). The development of gametophytes was inhibited in presence of high As concentration (8 mM) (Supplementary Table S1, online).

P. vittata plants hyperaccumulated As in fronds (Fig. 1b), in accordance with literature6. In particular, 60 days after As treatment the metalloid concentration in fronds (3350.00 ± 597.24 mg kg−1) was 21 times higher than in roots (157.14 ± 51.68 mg kg−1). Although being higher in fronds than in roots, the P content inside these fern tissues was unaffected by As exposure (Fig. 1). A significant increase of arsenic content was observed in As treated gametophytes (7106.67 ± 454.67 mg kg−1) (Fig. 1) while the phosphorus concentrations was unaffected by As exposure (Fig. 1).

Figure 1
figure 1

As and phosphorus concentration in P. vittata tissues.

Each value represents the mean of five replicates (n = 5) and its standard errors (±SE). Values followed by the same letter are not different, according to Fisher’s least significant difference test at a p ≤ 0.05. Capital letters and small letters indicated a comparison between different organs or treatments, respectively.

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In order to evaluate PvACR2 and Pv2.5–8 expression profiling in fronds, roots and gametophytes of P. vittata in response to As treatment, quantitative RT-PCR (qRT-PCR) (Fig. 2) was performed using EF-1b to compare the level of expression of PvACR2 and Pv2.5–8 genes. In qRT-PCR analysis, the mRNA level of PvACR2 gene in gametophytes and sporophyte fronds exposed or not to As was not altered, also Pv2.5–8 gene expression in sporophyte fronds was not affected by As treatment (Fig. 2a,c). On the contrary, the expression of the two genes in sporophyte roots and the Pv2.5–8 gene in gametophytes were boosted by As treatment (Fig. 2b,c).

Figure 2
figure 2

mRNA expression levels of PvACR2 and Pv2.5–8 in P. vittata fronds (a), roots (b) and gametophytes (c) by qRT-PCR. Green columns indicate controls whereas brown columns indicate As treatment. Each value represents the mean of three qRT-PCR replicates (n = 3) and its standard errors (±SE) repeated at least three times for each cDNA, from three different RNA extractions. Values followed by the same letter are not different, according to Fisher’s least significant difference test at a p ≤ 0.05.

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In order to assess the presence of arsenate reductase activity in fronds, roots and gametophytes of P. vittata, soluble proteins were extracted in mild conditions. Arsenate reductase activity was monitored by the coupled assay described by Ellis et al.5 with minor modifications. Arsenate reduction is coupled to NADPH oxidation via the reduction of oxidized GSH by GR, with GSH serving as the electron donor for arsenate reduction. Rate of NADPH oxidation was measured as decrease of optical density at 340 nm and was found to be minimal in the absence of frond, root and gametophytes protein extracts. Enzyme assay in the absence of arsenate was also performed as negative control. The decrease of NADPH absorbance at 340 nm observed in presence of the protein extract and the enzyme substrate (40 mM arsenate) was deducted by the absorbance reduction in presence of protein extract without arsenate. The contribution of the NADPH self-oxidation was valued by calculating the difference between the absorbance (∆Abs) observed in the mixture assay without the protein extract and the ∆Abs observed in presence of the protein extract. Arsenate reductase activity in fronds was inhibited by As exposure (Table 1), while in roots and gametophytes was unaffected by As treatment; in accordance with Ellis et al.5 , that reported a constitutive arsenate reductase activity in gametophytes.

Table 1 Arsenate reductase activity in P. vittata frond and root sporophytes treated or not once a week with 334 μM As and arsenate reductase activity in P. vittata gametophytes treated or not with 8 mM As.
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P. vittata sporophytes and gametophytes grown with or without As were used for the detection of PvACR2 and Pv2.5–8 in fern tissues using polyclonal antibodies. In particular, two recombinant proteins containing His6-tags, ACR2-His6 and Trx-His6-S-Pv2.5–8 were prepared in E. coli, purified under denaturing conditions and used as antigens in rabbit. The ability to identify PvACR2 and Pv2.5–8 and the title of polyclonal antibody products were compared with commercial monoclonal antibody anti-His6 tag signal (data not shown).

No bands were detected for PvACR2 protein in any of the fern tissues (data not shown), whereas anti-Pv2.5–8 antibodies detected a protein band in fronds, gametophytes and, to a minor extent, in roots (Fig. 3). The MW of this protein band was about 34.55 kDa, which is lower than the theoretical MW of Pv2.5–8 (45 kDa) (gi: 89243488), whose sequence is shown in Fig. 4. The signal intensity of the protein bands did not change with arsenic treatment in any of the tested tissues. Moreover, fronds and gametophytes showed similar signal intensity and it was higher than that observed in roots (Fig. 3).

Figure 3
figure 3

Immunoblot with anti-Pv2.5–8 antibody on P. vittata roots (lanes 1–2), fronds (lanes 3–4) treated or not once a week with 334 μM and gametophytes treated or not with 8 mM As (lanes 5–6).

Controls: lanes 1, 3, 5; arsenic treatment: lanes 2, 4, 6.

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Figure 4
figure 4

Pv2.5–8 amino acidic sequence (data from NCBI database): 435 amino acids and theoretical pI 9.31.

The Rhodanese homology domain (262–376) is yellow highlighted. The peptides identified by nanoLC-MS/MS analysis are in bold and underlined.

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In order to characterize this 34 kDa protein , the gel portion corresponding to the 25–50 kDa region, was transferred onto PVDF membranes, probed with anti-Pv2.5–8 polyclonal antibodies, excised, digested with trypsin and finally submitted to nanoLC-MS/MS analysis (Fig. 5). Four peptides corresponding to the Pv2.5–8 putative arsenate reductase were successfully identified and three of them partly cover the rhodanese homology domain (from aa 262 to aa 376) of this protein (gi: 89243488) (Fig. 4, Table 2). Then, to further characterize Pv2.5–8, 2-DE was performed. Interestingly, in fronds and gametophytes, Pv2.5–8 migrates as a series of spots having the same MW (about 35 kDa) of the protein band isolated on 1-DE gels, but with different isoelectric point (pI). More in detail, six spots with pI ranging from 8.3 to 8.9 were detected (Fig. 5). Fronds and gametophytes showed a similar protein pattern, regardless As treatment (data not shown). 2-DE was performed on root extracts too, but no spots were detected by antibodies.

Table 2 List of peptides derived from the immunoreactive (anti-Pv2.5–8) 1-DE bands and identified by MS/MS analysis.
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Figure 5
figure 5

P. vittata sporophytes frond proteins from control, stained with Blue-Silver (lane 1), immunodetected with anti-Pv2.5–8 polyclonal antibodies in 1-DE (lane 2) and 2-DE (panel).

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In order to detect PvACR2 in fern tissues, gametophyte and frond/root extracts were immuno-precipitated with anti-ACR2 polyclonal antibodies (Fig. 6). In all the immune-precipitated tissues anti-ACR2 polyclonal antibodies detected a band of about 14.4 kDa and this protein is present to a minor extent in roots, as observed for Pv2.5–8. This protein showed the same MW of the ACR2 recombinant protein.

Figure 6
figure 6

Immunoblot after immunoprecipitation with anti-ACR2 antibody from roots (lanes 1–2) and fronds (lanes 3–4) of P. vittata sporophytes treated or not with 334 μM As and gametophytes (lanes 5–6) treated or not with 8 mM As.

Controls: lanes 1, 3, 5; arsenic treatment: lanes 2, 4, 6. Recombinant ACR2 (lane 7) was used as a positive control of immunoprecipitation.

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Discussion

It’s well known that in As hyperaccumulator plants, such as P. vittata, arsenate (AsV) can be directly reduced to AsIII by arsenate reductase24. However, the plant organ where the AsV reduction occurs in P. vittata is still unknown. Some authors33,34 suggested that AsV is reduced to AsIII in the roots and subsequently transported to the fronds. Other works6,35 reported that AsV reduction to AsIII takes place directly in the fronds. More recently, AsV reduction has been observed in the rhizomes and in the pinnae but not in the roots of this fern36.

In our experimental conditions, sporophytes and gametophytes of P. vittata did not show macroscopic stress symptoms showing a more high tolerance of this fern to the metalloid. Arsenic treatment reduced the dry weight of the gametophytes and roots compared to controls. Consistently with previous works8,37 we found a higher amount of As in gametophytes and fronds of P. vittata. It has been postulated that in As hyperaccumulating plant species, As is not immobilized in roots, but it is transported through the xylem to the fronds6,38 and sequestered as free AsIII in the vacuole6,38. As recently demonstrated by Indriolo et al.39, the PvACR3 protein is involved in the vacuolar sequestration of AsIII in P. vittata.

Since AsV is a chemical analogue of phosphate, arsenic uptake by P. vittata occurs through phosphorus transporters28. Therefore, a competition between AsV and phosphate for the same transporter is expected to occur in plants grown in As polluted soils. However, according to Bona et al.8 that observed no differences in the roots, our results showed that the phosphorus content inside the plant organs was unaffected by As exposure, despite a high variability of the P content in each plant.

The PvACR2 mRNA levels were unaffected by exposure to different arsenic amounts (334 μM and 8 mM, respectively) in fronds and gametophytes of P. vittata. On the contrary, Pv2.5–8 mRNA levels increased in As-treated gametophytes, moreover an increase of both PvACR2 and Pv2.5–8 mRNA levels occurred in arsenic treated roots. The constitutive expression of PvACR2 in gametophytes of P. vittata is consistent with the findings of Ellis et al.5. However, the presence of Pv2.5–8 and PvACR2 mRNAs in fronds and roots and of Pv2.5–8 in gametophytes has never been reported before. These data suggest the involvement of these two proteins in the plant response to arsenic and can support, on the basis of mRNA levels up-regulation, the hypothesis that AsV reduction to AsIII occurs mainly in roots.

In a further experimental step, we detected and identified for the first time the two arsenate reductases (Pv2.5–8 and PvACR2) in both sporophytes and gametophytes of P. vittata at the protein level. The isolation of PvACR2 and Pv2.5–8 cDNAs from P. vittata tissues revealed the presence of PvACR2 in sporophytes and that of Pv2.5–8 in both gametophytes and sporophytes. Pv2.5–8 corresponds to a “predicted protein sequence of a putative arsenate reductase” submitted in 2006 to GenBank (Pv2.5–8, gi: 89243488) as an aldose reductase and rhodanese, similar to calcium binding protein (Rathinasabapathi et al. unpublished). The specific antibodies produced against Pv2.5–8 detected a protein band in fronds, gametophytes and, to a minor extent, in roots showing a molecular weight (about 35 kDa) lower than the theoretical MW of Pv2.5–8 (45 kDa). Since the 35 kDa protein band has been identified by MS/MS analysis as Pv2.5–8 (gi: 89243488), it’s likely that post-translational cleavage, not predictable at the transcription level, have occurred in fern tissues. More in detail, three of the identified peptides were part of the Rhodanese Homology Domain of this protein, suggesting that the catalytic portion of the protein remains unaltered, while a protein cleavage may occur in the N-terminal side of the amino acid sequence. Moreover, the detection of Pv2.5–8 on 2-DE gels using anti-Pv2.5–8 antibody as series of spots with the same molecular weight (35 kDa), but with different pIs, could be related to post-translational modifications, such as phosphorylation, where the addition of one or more phosphate group alters the protein pI leading to its acidification. The signal intensity of Pv2.5–8 in fronds and gametophytes was unaffected by As treatment. Moreover, the signal intensity of Pv2.5–8 in roots was lower than that detected in fronds and gametophytes. Despite the Pv2.5–8 mRNA levels in roots and gametophytes increased after As treatment, the protein Pv2–5–8 level did not change.

The immunoprecipitation of gametophyte and frond/root protein extracts revealed in each tissue the presence of a protein band of about 14 kDa, with the same MW of PvACR2, confirming the expression of ACR2 protein in P. vittata gametophyte and sporophyte. It is surprising that a so relevant enzyme for the arsenic metabolism (PvACR2) is expressed in such a low amount in P. vittata. Its presence in fronds and gametophytes seems to be higher than in roots, as observed also for Pv2.5–8.

Despite the mRNA level of both proteins increased in roots after As treatment, no difference was observed at the protein level; this suggests the presence of other molecular mechanisms that regulate the synthesis and the activity of these proteins. Arsenate reductase activity was detected in frond, root and gametophyte extracts. Whereas the activity did not change in roots and in gametophytes following As treatment, higher AsV reductase activity was detected in frond of control plants compared to the arsenic treated ones. Multiple enzymes have been shown to exhibit AsV reductase activity2; these included glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase and phosphoglycerate kinase that in P. vittata fronds treated with As showed a lower protein expression level8 and probably a less reductase activity. The enzymatic activity and its trend in relation to arsenic exposure were consistent with the findings of Liu et al.34, who noticed a slight decrease of arsenate reductase activity in fronds along with the increase of arsenic added in soil up to 100 ppm, followed by an increase of the enzyme activity with arsenic concentrations higher than 100 ppm. This observation seems to be in contradiction with the detection of higher amounts of both Pv2.5–8 and PvACR2 proteins in fronds than in roots, even if unaffected by arsenic treatment. Moreover, an increase of PvACR2 and Pv2.5–8 transcripts after arsenic exposure was observed in roots. We can speculate that roots are not the organs for arsenic storage in P. vittata, consequently a constitutive high amount of arsenate reductase is not required, but its production together with a high protein turnover rate, is induced after arsenic exposure to allow the prompt arsenate reduction and transfer to fronds. On the contrary, fronds are the elected organs for the storage of arsenic: therefore, the maintenance of a constitutive pool of arsenate reductase in fronds is essential in case of re-oxidation of AsIII to AsV, which can occur in senescent leaves, where reductase activities usually decrease while oxidase activities increase40.

In conclusion, the constitutive expression of both P. vittata arsenate reductase proteins in fronds, roots and gametophytes, suggests that the reduction of arsenate is carried out by a system of arsenate reductases with the support of other enzymes having metabolic essential functions. These conclusions are sustained by other works showing the involvement of a cytosolic triosephosphateisomerase (TPI) in arsenate reduction in P. vittata41, the activation of multiple pathways to tolerate As8,9,42 and the newly identified arsenate reductase in A. thaliana19,20. Overall the hyperaccumulating fern P. vittata displays different molecular strategies for As tolerance in roots and fronds, which need to be considered for the characterization of its phytoremediation potential.

Methods

Plant material

P. vittata spores were sterilized as described by Trotta et al.43. After one month, half of the gametophytes were transferred to sterile polyethylene boxes for sporophyte production. The remaining gametophytes were transferred to 9 cm Petri dishes containing 15 ml liquid MS medium (Murashige and Skoog 1962)44 added with 2% sucrose, with or without arsenate 8 mM (supplied as Na2HAsO4·7H2O) (Sigma-Aldrich, MO, USA) and maintained for 10 days in a growth chamber with 16/8 h light/dark photoperiod, 150 μmol m−2 s−1 light irradiance and 24/20 °C thermoperiod. Then, the gametophytes were washed and weighed. Part of the samples was dried at 60 °C for 72 h and used for the determination of arsenic (As) and phosphorus (P) concentration, while the rest was frozen in liquid nitrogen and stored at −80 °C.

The sporophyte experiment was performed according to Bona et al.8,9.

Arsenic and phosphorus concentration

The As and P concentrations were measured both in roots and in fronds. 0.5 g dry weight of each sample were digested in 6 ml of 65% nitric acid (Sigma-Aldrich) using a MARS 5 microwave oven (CEM, North Carolina, USA). Digested samples were analysed by inductively coupled plasma-optical emission (IRIS Advantage ICAP series DUO HR, Thermo Jarrell Ash, Franklin, USA) and inductively coupled plasma-MS (Plasma QUAD 3, VG Elemental Europe, Cedex, France). Certified standards of analysed metals and acid blanks were run. The As and P concentrations were expressed as mg kg−1.

RNA extraction and quantitative RT-PCR

Total RNA was extracted from 500 mg of P. vittata fronds, roots and gametophytes, combining CTAB method45 and NucleoSpin RNA Plant kit (Macherey-Nagel, Düren, Germany). Briefly, 500 mg of tissue powdered in liquid nitrogen were added to 500 μl of extraction buffer (2% CTAB, 2% polyvinylpyrrolidone (PVPP) MW 4000, 100 mM Tris-HCl pH 8.0, 20 mM EDTA, 1.4 M NaCl and 2% β-mercaptoethanol). An equal volume of chloroform-isoamyl alcohol (24:1), previously heated for 10 min at 60 °C, was added to the mixture. The suspension was then centrifuged at 16000 g for 8 min at 4 °C. RNA was precipitated with isopropanol for 60 min at −20 °C and cleaned using the Nucleospin RNA plant Kit. RNA purity, quantity and integrity were assessed46. 1–2 μg of total RNA from P. vittata tissues were treated with Dnase I (Sigma-Aldrich) and reverse transcripted to cDNAs using oligo (dT)18 primer and RevertAid H Minus First Strand cDNA synthesis kit (Fermentas, Canada). Then 1 μl of sample was diluted in a DEPC-treated water solution containing 0.02 U μl−1 of Taq DNA polymerase (Finnzymes, Finland), 10× PCR buffer (containing 15 mM MgCl2) (Finnzymes), 500 μM of dNTPs (125 μM each dNTP) and 500 nM of the PvACR2 or Pv2.5–8 forward and PvACR2 or Pv2.5–8 reverse primers (Supplementary Table S2, online) and amplified by PCR in a thermocycler (Techne, Bibby Scientific, Italy). PCR conditions were 5 min at 94 °C, 30–33 cycles of 1 min at 94 °C, 30–33 cycles of 1 min at 55 °C, 30–33 cycles of 1 min and 30 s at 72 °C, 10 min at 72 °C. The resulting fragments were cloned into pCR4-TOPO vector using the TOPO TA Cloning Kit for Sequencing (Invitrogen, CA, USA) sequenced by BMR Genomics (Padova, Italy) using primer T3 and compared to the sequences of PvACR2 and Pv2.5–8 available in GenBank by using the BLASTn program47.

Quantitative RT-PCR (qRT-PCR) of PvACR2 and Pv2.5–8 was performed in a multiplex Taqman assays using P. vittata elongation factor-1b (EF-1b) as constitutive internal standard5. Probes dual-labeled and primer pairs (Supplementary Table S2, online) were designed using Beacon Designer v3.0 (Premier Biosoft International, Inc.). cDNA was amplified in a CFX384 Real-Time PCR (Bio-Rad) using 0.3 μM each primer, 0.1 μM each probe iQTM Multiplex Powermix (Bio-Rad) according to the manufacturer’s instructions for the triplex protocol, in a final volume of 10 μL. For all Taqman assays the thermal protocol was: 3 min at 95 °C, followed by 46 cycles of 15 s at 95 °C and 20 s at 59 °C. Relative expression data were geometrically normalized to EF-1b. qRT-PCR was performed on three different biological replicates and repeated at least three times for each cDNA.

Protein expression, purification and antibody production

The PvACR2 and Pv2.5–8 reading frames were amplified by PCR with primers adding a NcoI and XhoI site to the 5′ and 3′ ends of the fragment, respectively (Supplementary Table S2, online). PCR conditions for both genes were 5 min at 94 °C, 30 cycles of 1 min at 94 °C, 30 cycles of 1 min at 58 °C, 30 cycles of 1 min and 30 s at 72 °C, 10 min at 72 °C. The fragments were digested with NcoI and XhoI, then the Pv2.5–8 fragment was ligated into pET32a (Novagen, United Kingdom) in frame at 5′ with thioredoxin, S-tag and His6-tag, creating plasmid pET-Pv2.5–8 and the PvACR2 fragment was ligated into pET20b (Novagen) in frame at 5′ with pelB signal sequence and at 3′ with His6-tag, creating plasmid pET-PvACR2.

For protein expression, E. coli BL21DE3 cells (Stratagene, Canada) transformed with pET-PvACR2 or pET-Pv2.5–8 were grown in at 37 °C in Luria-Bertani medium containing 50 μg ml−1 ampicillin. At 0.5 of absorbance at 600 nm, isopropylthio-β-galactoside (IPTG) was added to a final concentration of 0.4 mM and the cell cultures were incubated for 4 h at 37 °C. Recombinant proteins were purified using the Ni-NTA Agarose resin (QIAGEN, Italy) under denaturing conditions. Recombinant PvACR2 and Pv2.5–8 were identified by SDS-PAGE46, dialyzed against 1000 volumes of 100 mM NaH2PO4, 10 mM Tris, 2.5 M urea, pH 7.4, concentrated using Microcon YM-3 (Millipore, Germany) and quantified by Bradford method48. Antigenic preparations were emulsified with complete Freund adjuvant (MP BioMedicals, Illkirch, France) and injected into NZW rabbits. A second booster injection was given 30 days later using polyA-polyU as adjuvant. At 2-wk intervals, bleeding of the rabbits was performed according to standard procedure49. Different serum dilutions were tested against recombinant proteins separated by SDS-PAGE and transferred onto a nitrocellulose membrane (0.2 μm or 0.45 μm). For immunodetection, membranes were saturated with PBS/BSA 5% and probed with anti-His6 monoclonal antibodies (GE-Healthcare, Cologno Monzese, Italy) or with the sera of rabbits immunized with the two P. vittata recombinant proteins. Immunodetection was performed using either chemiluminescence reaction or chromogenic reaction.

Protein extraction, Two-dimensional gel electrophoresis (2-DE), nanoLC-MS/MS analysis

Proteins were extracted from gametophytes and sporophyte tissues according to Bestel-Corre et al.50 with some modifications and 2-DE separated as reported in Bona et al.8,9.

In parallel, frond and root extracts were also separated by monodimensional SDS-PAGE (1-DE). 1-DE and 2-DE gels were stained with Blue Silver colloidal Coomassie, according to Candiano et al.51, or transferred onto nitrocellulose or PVDF membranes. The development of membranes from was performed as described above, using rabbit polyclonal antibodies against PvACR2 and Pv2.5–8 as the primary antibody and an alkaline phosphatase-conjugated ‘anti-rabbit’ serum as secondary antibody (Sigma-Aldrich). The antibody reaction was detected by chromogenic reaction. Images were acquired with GS-710 densitometer (BioRad).

Nine replicates of the immuno-detected 1-DE bands were excised and trypsin digested as reported in detail in Bona et al.8. Digested samples were nanoLC-MS/MS analysed using a QSTAR XL instrument (Applied Biosystems, CA, USA) coupled with nano-flow LC system (Dionex, Amsterdam, The Netherlands). Protein identification was performed using the MASCOT search engine52.

PvACR2 immunoprecipitation from plant tissues

About 600 μg of gametophyte and sporophyte protein extracts were dialysed against 5 volumes of NP-40 buffer. 1 μl of rabbit serum was added to each tube and incubated ON at 4 °C. ProteinA-Sepharose CL 4B resin (GE Healthcare) was resuspended in 1 vol of water, centrifuged at 3000 g at 4 °C for 6 min, washed twice and resuspended in water to a 1:1 ratio. 50 μl of protein A-Sepharose CL 4B were activated, added to each sample and incubated at 4 °C for 60 min. Samples were then centrifuged (3000 g, 6 min, 4 °C), the pellet was washed twice with 300 μl of PBS. The pellet was then resuspended in 40 μl of 1-DE Laemmli buffer containing β-mercaptoethanol, denaturated for 10 min at 95 °C and stored at −20 °C.

Arsenate reductase assay of P. vittata extracts

Gametophytes, fronds and roots of P. vittata were powdered in liquid nitrogen, resuspended in five volumes of extraction buffer (50 mM MES/MOPS pH 6.5, 0.3 M KCl, 10 mM β-mercaptoethanol, protease inhibitor cocktail 1%, PVPP 0.033 g ml−1, glycerol 5%) and centrifuged at 1,860 g for 15 min at 4 °C. The supernatant was centrifuged at 20,000 g for 60 min at 4 °C and concentrated using Amicon Ultra-4 (Millipore) with a cutoff of 10 kDa. Arsenate reductase activity was measured using a coupled assay as described by Ellis et al.5. The assay mixture consisted of 50 mM MES/MOPS pH 6.5, 300 mM NaCl, 0.1 mg ml−1 BSA, 1 mM GSH, 250 μM NADPH, 1 U yeast glutathione reductase (GR), 70 μg of total protein extract, 40 mM sodium arsenate (Na2HAsO4 7H2O) or an equal volume of water. The assay was carried out in a final volume of 1.5 ml at room temperature. Reductase activity was monitored for 90 min at 340 nm by a DU 800 spectrophotometer (Beckman, CA, USA). The nmoles of oxidized NADPH were calculated using a molar extinction coefficient of 6,200 M−1cm−1.

Statistical analysis

Statistical analysis was performed with StatView 4.5 software (Abacus Concepts, CA, USA). ANOVA test, followed by a post-hoc F test with p ≤ 0.05 as cut off.

Additional Information

How to cite this article: Cesaro, P. et al. The arsenic hyperaccumulating Pteris vittata expresses two arsenate reductases. Sci. Rep. 5, 14525; doi: 10.1038/srep14525 (2015).