Vacuolar Phosphate Transporter 1 (VPT1) Affects Arsenate Tolerance by Regulating Phosphate Homeostasis in Arabidopsis

Vacuolar Phosphate Transporter 1 (VPT1) Affects Arsenate Tolerance by Regulating Phosphate... Abstract Arsenate [As(V)] is toxic to nearly all organisms. Soil-borne As(V) enters plant cells mainly through the plasma membrane-localized phosphate (Pi) transporter PHT1 family proteins due to its chemical similarity to Pi. We report here that VPT1, a major vacuolar phosphate transporter which contributes to vacuolar Pi sequestration, is associated with As(V) tolerance in Arabidopsis. vpt1 mutants displayed enhanced tolerance to As(V) toxicity, whereas plants overexpressing VPT1 were more sensitive to As(V) as compared with the wild-type plants. Measurements of arsenic content indicated that vpt1 mutants accumulated less arsenic and, in contrast, up-regulating VPT1 expression contributed to higher levels of arsenic accumulation in plants. To examine further how VPT1 may modulate arsenic contents in plants, we surveyed the expression patterns of all the PHT1 family members that play roles in As(V) uptake, and found that many of the PHT1 genes were down-regulated in the vpt1 mutant as compared with the wild type under Pi-sufficient conditions, but not when Pi levels were low in the medium. Interestingly, As(V) sensitivity assays indicated that As(V) resistance in vpt1 mutants was prominent only under Pi-sufficient but not under Pi-deficient conditions. These results suggest that under Pi-sufficient conditions, loss of VPT1 leads to elevated levels of Pi in the cytosol, which in turn suppressed the expression of PHT1-type transporters and reduced accumulation of arsenic. Introduction Oxidized arsenic species are identified as a non-threshold class-I human carcinogen (Salt 2017). Arsenic contamination has become increasingly severe in soil and water resources due to industrial wastewater discharge and application of pesticides in agriculture (Moreno-Jimenez et al. 2012). Arsenic exposure through drinking water and food negatively impacts human health, leading to increased risk of cancer and other diseases (Bundschuh et al. 2012, Joseph et al. 2015). Soil-borne arsenic can accumulate in the plant root system and inhibit cell division and elongation (Garg and Singla 2011). When transported to the upper parts of a plant, arsenic damages the chloroplast structure and causes a decline in plant growth (Finnegan and Chen 2012). Arsenic exists in two inorganic forms, namely arsenate [As(V)] and arsenite [As(III)]. As(V) is a chemical analog of phosphate (Pi), and may disrupt Pi-dependent aspects of metabolism (Raghothama 1999, Tu and Ma 2003, Wu et al. 2011). As(III) binds to the sulfhydryl group of cysteine in many proteins with a high affinity, and then severely affects protein structure, leading to adverse effects on many metabolic processes (Kitchin and Wallace 2006). Arsenic in the soil is absorbed by plants and may accumulate in edible parts such as fruits and seeds (Williams et al. 2007, Sohn 2014). Therefore, understanding the arsenic transport mechanisms in plants is critical to development of genetic tools that limit arsenic accumulation in crop species. As(III) mainly exists in anaerobic soil such as paddy fields which are used for cultivating rice (Xu et al. 2008). Studies indicate that As(III) is transported into plants mainly through NIP (nodulin 26-like intrinsic proteins) family proteins (Ma et al. 2008, Xu et al. 2015). Disruption of NIP1;1 function significantly enhances the resistance to arsenite in Arabidopsis (Kamiya et al. 2009). NIP1;3, another NIP family member, is responsible for root to shoot translocation of As(III) (Xu et al. 2015). When As(III) is transported into cells, it can be chelated by phytochelatins (PCs) as As(III)–PCs, and then this complex is sequestrated by the vacuole through ABCC1/2 (C-type ABC transporters 1 and 2) (Song et al. 2010). As(V) is a primary arsenic form in aerobic soils (Moreno-Jimenez et al. 2012), and accounts for 5–20% of total soluble arsenic in anaerobic soil (Khan et al. 2010, Zhang et al. 2015). Due to the chemical similarity to Pi, As(V) enters plant cells mainly through the high-affinity Pi transport systems, especially PHT1 (phosphate transporter1)-type transporters (Shin et al. 2004, Catarecha et al. 2007). Thus, mutations of genes encoding these transporters may reduce As(V) uptake and sensitivity to As(V) stress in plants. For instance, loss of function of PHT1;1 and PHT1;4, the major Pi transporters involved in Pi uptake, confers enhanced As(V) tolerance in Arabidopsis (Shin et al. 2004). Loss of function of PHT1;9, another plasma membrane-localized phosphate transporter, reduces arsenic content in plants under As(V) toxic conditions, while overexpressing PHT1;9 results in enhanced arsenic accumulation (Remy et al. 2012). Similarly, the knockout of OsPT8, a plasma membrane-localized PHT family member in rice, reduces the As(V) content to about 50% of that of control plants (Wang et al. 2016), whereas its overexpression causes overaccumulation of arsenic in plants (Wu et al. 2011). Moreover, regulatory proteins governing the expression of PHT1-type genes are also shown to play a role in As(V) tolerance. The WRKY6 transcription factor restricts arsenate uptake efficiency by inhibiting the expression of PHT1;1 (Castrillo et al. 2013). Loss of phosphate transporter traffic facilitator1 (PHF1), an endoplasmic reticuulum (ER) protein that facilitates PHT1;1 targeting to the plasma membrane, also enhances As(V) tolerance (Gonzalez et al. 2005). In contrast, up-regulation of OsPHR2, a MYB transcriptional factor gene that activates the expression of OsPT genes, would contribute to As(V) uptake (Wu et al. 2011). Therefore, plasma membrane-localized PHT1-type Pi transporters are closely related to arsenic accumulation in plants. Furthermore, Pi status in the soil and in plants also contributes to the extent of As(V) toxicity. It has been shown that low Pi conditions exacerbate plant sensitivity to As(V), while Pi application to the soil enhances As(V) tolerance in plants (Tu and Ma 2003, Catarecha et al. 2007). Taken together, a better understanding of the cross-talk between Pi balance and As(V) tolerance should contribute to the knowledge base for improving As(V) tolerance in plants. Studies so far have been focused on the relevance of plasma membrane-localized Pi transporters (PHT1s) for their role in As(V) uptake as described above. However, the endomembrane-localized Pi transporters and their contribution to As(V) toxicity have not been addressed. In particular, the vacuole works as the major pool of inorganic phosphorus in the plant cells and plays a key role for Pi homeostasis maintaining in plants (Pratt et al. 2009). Recent findings showing that vacuolar phosphate transporter 1 (VPT1; or PHT5; 1) as the major Pi transporter responsible for vacuolar Pi accumulation (Liu et al. 2015, T.Y. Liu et al. 2016), providing an opportunity for examining its potential role in As(V) toxicity. In this study, we found that vpt1 mutants displayed enhanced tolerance to As(V) toxicity specifically under Pi-sufficient conditions possibly by inhibiting the expression of PHT1-type transporters. Results and Discussion vpt1 mutants display enhanced As(V) tolerance Phosphate transporters (such as PHT1 family proteins) are critical in fine-tuning Pi homeostasis in plant cells, which affect the sensitivity to As(V) stress (Li et al. 2016). VPT1 has recently been identified as a major vacuolar phosphate transporter, and disruption of its function alters Pi homeostasis, with more Pi arrested in the cytosol (Liu et al. 2015, J. Liu et al. 2016). As Pi is closely related to As(V) tolerance in plants, we expected that VPT1 may affect As(V) tolerance in plants. To test this hypothesis, we compared two Arabidopsis mutant lines containing T-DNA insertional alleles of the VPT1 gene (vpt1-1 and vpt1-2) with wild-type plants under various concentrations of As(V) in the culture medium. After germination in normal half-strength Murashige and Skoog (1/2 MS) medium for 4 d, the plants were transferred to medium containing 0, 250, 500 or 700 µM As(V). As shown in Fig. 1A, the growth of all seedlings was severely inhibited by increasing As(V) concentrations. However, vpt1 mutant plants consistently displayed more tolerance to these As(V) treatments as compared with the wild-type plants (Fig. 1B). For instance, the fresh weight of the vpt1 mutant was significantly higher than that of the wild type once the As(V) concentration reached 250 µM and beyond. At 700 µM, As(V) severely inhibited the growth of wild-type seedlings that displayed a darker color, reflecting elevated levels of anthocyanin [Fig. 1A;Supplementary Fig. S1]. In addition to larger cotyledons (Supplementary Fig. S1), vpt1 mutants also had longer roots than the wild type (Fig. 1C). The length of vpt1 roots was nearly 50% longer than that of the wild-type plants when grown on the culture medium with 700 µM As(V). Fig. 1 View largeDownload slide vpt1 single mutants are less sensitive to toxic levels of As(V). (A) Wild-type and vpt1 seedlings under different As(V) conditions. Four-day old seedlings from a 1/2 MS plate were transferred onto plates with 0 µM (I), 250 µM (II), 500 µM (III) and 700 µM (IV, V) As(V) and cultured for 7 d. Scale bars represent 1 cm (I, II, III and IV) and 1 mm (V). The single seedling fresh weight and primary root length (B, C) were measured for phenotyping. The results were reproducible in three independent experiments. Data are the mean ± SD. n = 8. Asterisks represent a significant difference between vpt1 mutant lines and the wild type under the same culture conditions. Student’s t-test, *P < 0.05, **P < 0.01. Fig. 1 View largeDownload slide vpt1 single mutants are less sensitive to toxic levels of As(V). (A) Wild-type and vpt1 seedlings under different As(V) conditions. Four-day old seedlings from a 1/2 MS plate were transferred onto plates with 0 µM (I), 250 µM (II), 500 µM (III) and 700 µM (IV, V) As(V) and cultured for 7 d. Scale bars represent 1 cm (I, II, III and IV) and 1 mm (V). The single seedling fresh weight and primary root length (B, C) were measured for phenotyping. The results were reproducible in three independent experiments. Data are the mean ± SD. n = 8. Asterisks represent a significant difference between vpt1 mutant lines and the wild type under the same culture conditions. Student’s t-test, *P < 0.05, **P < 0.01. Arsenic is present mainly as As(V) and As(III) that are both toxic to plants. Several studies suggested that As(V) in the cytosol could be rapidly reduced to As(III) by arsenate reductase (AR) (Bleeker et al. 2006, Duan et al. 2007, Salt 2017). In fact, As(III) is much more toxic to plant cells than As(V). Studies indicated that micromolar levels of As(III) can be inhibitory to plant growth (Ji et al. 2017). To test if enhanced As(V) tolerance in vpt1 was a result of altered sensitivity to As(III) derived from As(V), we cultured the seedlings on 1/2 MS medium with various concentrations of As(III). The experimental results indicated that As(III) was indeed more toxic to plants and 5 µM already severely inhibited the growth of Arabidopsis plants (Fig. 2A). However, the vpt1 mutant and the wild-type plants showed similar sensitivity to As(III), as measured by root length and fresh weight (Fig. 2B, C). Additionally, we surveyed the expression patterns of genes involved in As(III) resistance, including ACR2, ABCC1, ABCC2 and NIP1;1 (Ma et al. 2008, Kamiya et al. 2009, Song et al. 2010). The quantitative real-time PCR (qPCR) data indicated that the expression levels of these genes were similar in vpt1 and wild-type plants (Supplementary Fig. S2). This suggests that loss of function of VPT1 specifically affects the sensitivity to As(V) in Arabidopsis. Fig. 2 View largeDownload slide VPT1s are not related to As(III) resistance. (A) Various genotypes showed no As(III) resistance. Four-day old seedlings of various genotypes from a 1/2 MS plate were transferred on plates with 2, 5 and 9 µM As(III) separately and cultured for 7 d. The fresh weight and root length are presented in (B) and (C). Data are the mean ± SD, n = 4. The results were reproducible in three independent experiments; scale bars indicate 2 cm. Fig. 2 View largeDownload slide VPT1s are not related to As(III) resistance. (A) Various genotypes showed no As(III) resistance. Four-day old seedlings of various genotypes from a 1/2 MS plate were transferred on plates with 2, 5 and 9 µM As(III) separately and cultured for 7 d. The fresh weight and root length are presented in (B) and (C). Data are the mean ± SD, n = 4. The results were reproducible in three independent experiments; scale bars indicate 2 cm. VPT1 overexpression enhances arsenate sensitivity in Arabidopsis To examine further the function of VPT1 in plant As(V) stress, we overexpressed VPT1 in wild-type Arabidopsis. The Pi content measurements showed that overexpressing VPT1 leads to increased Pi accumulation in Arabidopsis (Supplementary Fig. S3), which is also indicated by other studies (Liu et al. 2015, T.Y. Liu et al. 2016). We selected two transgenic lines (OV1-1 and OV1-2) with higher levels of VPT1 transcripts for phenotyping. The As(V) sensitivity assays showed that OV1-1 and OV1-2 seedlings were more sensitive to As(V) stress as compared with the wild-type seedlings, while the vpt1 single mutant displayed enhanced tolerance to As(V) as described earlier (Fig. 3A). For instance, 400 µM As(V) inhibited root growth and reduced the fresh weight of the seedlings in all genotypes, but OV1 plants were more affected than the wild type and the vpt1 mutant (Fig. 3B, C). There appeared to be a positive correlation between VPT1 expression level and As(V) sensitivity (Supplementary Figs. S3, S4). For instance, OV1-1 and OV1-2 seedlings showed higher levels of VPT1 mRNA (about 21 times higher than the wild type), correlated with shorter roots and lower fresh weight under As(V) stress, whereas OV1-3 seedlings had a lower VPT1 mRNA level (about six times higher than the wild type) as compared with OV1-1/OV1-2 and were less sensitive to As(V) toxicity (Supplementary Figs. S3, S4). The OV1-4 seedling exhibited the least sensitivity to As(V) among the VPT1-overexpressing lines and its VPT1 expression level was about twice as high as that in wild-type seedlings. Fig. 3 View largeDownload slide VPT1 modulates sensitivity to arsenate in Arabidopsis. (A) Phenotypes of various genotypes under As(V) toxic conditions. Three-day-old seedlings from a 1/2 MS plate were transferred to a plate with 400 µM As(V) and cultured for 6 d. Scale bars represent 1 cm. Primary root length (B) and the single seedling fresh weight (C) were measured for phenotyping. The results were reproducible in three independent experiments. Data are the mean ± SD, n = 8. Different letters indicate a significant difference among various seedling lines under the same culture conditions. Student’s t-test, **P < 0.01. (D) VPT1 is related to As(V) accumulation in Arabidopsis. Different 8-day-old genotypes were transferred to medium with 400 µM As(V) and sampled at 0, 1, 2, 3 and 4 d for arsenic measurements. Data are the mean ± SD, n = 4. Fig. 3 View largeDownload slide VPT1 modulates sensitivity to arsenate in Arabidopsis. (A) Phenotypes of various genotypes under As(V) toxic conditions. Three-day-old seedlings from a 1/2 MS plate were transferred to a plate with 400 µM As(V) and cultured for 6 d. Scale bars represent 1 cm. Primary root length (B) and the single seedling fresh weight (C) were measured for phenotyping. The results were reproducible in three independent experiments. Data are the mean ± SD, n = 8. Different letters indicate a significant difference among various seedling lines under the same culture conditions. Student’s t-test, **P < 0.01. (D) VPT1 is related to As(V) accumulation in Arabidopsis. Different 8-day-old genotypes were transferred to medium with 400 µM As(V) and sampled at 0, 1, 2, 3 and 4 d for arsenic measurements. Data are the mean ± SD, n = 4. A recent study indicates that As(V) inhibits root hair growth (Forino et al. 2012). We thus examined the effect of As(V) on root hair growth in plants of different genotypes (Supplementary Fig. S5). Under control condition without As(V), all plants had similar root hairs of 200–600 µm long (Supplementary Fig. S5A, B). In the presence of 400 µM As(V) in the medium, the length of root hairs was in the range of 50–200 µm in OV1 plants, 100–300 µm in wild-type seedlings and 200–500 µm in the vpt1 mutant plants (Supplementary Fig. S5A, C). This supports the notion that overexpression of VPT1 causes hypersensitivity to As(V) in plants. Given that As(V) uptake directly affects the sensitivity to As(V) toxicity in plants (Shin et al. 2004, Wu et al. 2011), we suspected that the amount of As(V) absorbed by plants of different genotypes (the wild type, vpt1 mutant and VPT1-overexpressing lines) may be different and would directly correlate with inhibition of plant growth (Shin et al. 2004, Wu et al. 2011). After 8-day-old seedlings were transferred to medium with 400 µM As(V) and cultured for a further 4 d, we measured arsenic contents of different plants and found that vpt1-1 mutant plants contained less arsenic while OV1-1 seedlings accumulated more arsenic, as compared with the wild type (Fig. 3D). On day 4 after As(V) treatment, the arsenic content of the vpt1-1 mutant was about 23% less than in the wild type, while OV1-1 plants contained 35% more arsenic than wild-type seedlings. This is consistent with the extent of toxicity observed in these plants. VPT1-associated As(V) sensitivity depends on Pi status In general, inhibition of As(V) uptake will decrease arsenic accumulation in plants (Remy et al. 2012, Wang et al. 2016). As(V) could be transported by Pi transporters due to its chemical similarity to phosphate, and plasma membrane-localized Pi transporters are known to be responsible for arsenate acquisition. However, VPT1 is a vacuolar phosphate transporter (Liu et al. 2015, T.Y. Liu et al. 2016) and it is not known how VPT1 may affect arsenic accumulation in Arabidopsis. In particular, As(V) is largely converted into As(III) in the cytosol and we do not expect a role for VPT1 in vacuolar sequestration of As(V) (Bleeker et al. 2006, Duan et al. 2007, Salt 2017). Our previous study showed that loss of VPT1 leads to higher levels of Pi in the cytosol (Liu et al. 2015), leading to down-regulation of PHT1;1 and PHT1;4 under Pi-sufficient conditions. A number of previous studies have shown that reduced expression levels of PHT1 genes would enhance As(V) tolerance in plants (Shin et al. 2004, Catarecha et al. 2007, Remy et al. 2012, Wang et al. 2016). Therefore, lower expression levels of PHT1 family members may account for reduced uptake of As(V) in the vpt1 mutant. We analyzed the expression patterns of PHT1 family members in the vpt1 mutant and wild-type plants, and found that many of the PHT1 family genes, including PHT1;1, PHT1;2, PHT1;3, PHT1;4, PHT1;8 and PHT1;9, were significantly down-regulated in the vpt1 mutant as compared with wild-type plants under a Pi-sufficient condition (0.625 mM) (Fig. 4A). Furthermore, we analyzed the expression levels of the PHT1 gene in the vpt1 mutant grown under various Pi conditions (i.e. 20, 50, 200 and 625 µM, and 2 mM). We found that PHT1 genes in the vpt1 mutant were also induced as the Pi concentration in the medium decreased (Supplementary Table S2). When the seedlings grew under a Pi-deficient condition (20 µM Pi), the expression levels of PHT1 genes (i.e. PHT1;1, PHT1;3, PHT1;4, PHT1;7, PHT1;8 and PHT1;9) in the vpt1 mutant were comparable with those in the wild type (Fig. 4B). Fig. 4 View largeDownload slide The expression of PHT1 family genes is altered in the vpt1 mutant. (A and B) The expression patterns of PHT1 family members in the wild type and the vpt1 single mutant. Ten-day-old seedlings that were cultured in Pi-sufficient (0.625 mM) and Pi-deficient (20 µM) medium were obtained for determination of the PHT1 gene expression level. Wild-type seedlings were used as control; values were normalized to UBQ10. Data are the mean ± SD, n = 4. Statistical analysis was conducted between wild-type (Col-0) and vpt1 mutant plants by using Student’s t-test (*P < 0.05, **P < 0.01). Fig. 4 View largeDownload slide The expression of PHT1 family genes is altered in the vpt1 mutant. (A and B) The expression patterns of PHT1 family members in the wild type and the vpt1 single mutant. Ten-day-old seedlings that were cultured in Pi-sufficient (0.625 mM) and Pi-deficient (20 µM) medium were obtained for determination of the PHT1 gene expression level. Wild-type seedlings were used as control; values were normalized to UBQ10. Data are the mean ± SD, n = 4. Statistical analysis was conducted between wild-type (Col-0) and vpt1 mutant plants by using Student’s t-test (*P < 0.05, **P < 0.01). In parallel, we performed an As(V) tolerance assay under various Pi levels (Fig. 5A). On the culture medium with a low level of Pi (20 µM), the vpt1 mutant and the wild-type plants were similarly sensitive to As(V). As the Pi concentration was elevated, vpt1 became less sensitive to As(V) stress as compared with the wild type. When the Pi concentration reached 0.625 mM (sufficient Pi condition), the root length and fresh weight of the vpt1 mutant were about 31% and 45% more than in wild-type plants (Fig. 5B–E). However, under high Pi conditions (2 mM), the sensitivities to As(V) in vpt1 and the wild type were difficult to assess because vpt1 is more sensitive to high Pi conditions (Fig. 5A, E) (Liu et al. 2015). Moreover, arsenic content in the vpt1 mutant was lower than in the wild type when the Pi concentration in the medium was >200 µM, whereas such a difference disappeared under lower Pi conditions (<50 µM) (Supplementary Fig. S6). Thus, the sensitivity to As(V) stress in the vpt1 mutant depends on the Pi status, and altered cytosol Pi homeostasis might be related to the enhanced As(V) tolerance in the vpt1 mutant. Fig. 5 View largeDownload slide The vpt1-related resistance to As(V) depends on Pi status. (A) As(V) sensitivity assays under various Pi conditions. Three-day old seedlings were transferred to 1/2 MS medium plates with various concentrations of Pi (i.e. 20, 50, 200 and 625 µM, and 2 mM) and cultured for 7 d. As(V) at 400 µM was applied for As(V) sensitivity assays; scale bars represent 2 cm. The root length (B, C) and fresh weight (D, E) were used for qualification of As(V) sensitivity assays results. Data are the mean ±SD, n = 8. Student’s t-test (*P < 0.05, **P < 0.01). Fig. 5 View largeDownload slide The vpt1-related resistance to As(V) depends on Pi status. (A) As(V) sensitivity assays under various Pi conditions. Three-day old seedlings were transferred to 1/2 MS medium plates with various concentrations of Pi (i.e. 20, 50, 200 and 625 µM, and 2 mM) and cultured for 7 d. As(V) at 400 µM was applied for As(V) sensitivity assays; scale bars represent 2 cm. The root length (B, C) and fresh weight (D, E) were used for qualification of As(V) sensitivity assays results. Data are the mean ±SD, n = 8. Student’s t-test (*P < 0.05, **P < 0.01). In summary, the above results have connected Pi homeostasis and As(V) toxicity through the study of VPT1, a tonoplast-localized phosphate transporter responsible for vacuolar Pi sequestration (Liu et al. 2015, T.Y. Liu et al. 2016). The vacuole is the major phosphate storage space in plant cells, and its Pi concentration often reaches >10 mM, whereas for the cytosol, its Pi concentration is only about 60 μM (Pratt et al. 2009). When Pi is at sufficient or excess levels, the vacuole sequesters extra Pi that becomes remobilized under Pi-deficient conditions. Therefore, VPT1 is critical for Pi homeostasis in plants for its role in vacuolar Pi accumulation. As a result, the vpt1 mutant is less capable of adapting to changing Pi status in the environment. Loss of VPT1 would also severely reduce vacuolar Pi content and cause elevated Pi levels in the cytosol. The altered intracellular Pi homeostasis in vpt1 mutant cells contributes to altered expression of PSR (phosphate starvation response) genes (Liu et al. 2015, T.Y. Liu et al. 2016), including those encoding PHT1 family Pi uptake transporters which are also responsible for As(V) uptake (Shin et al. 2004, Catarecha et al. 2007, Wu et al. 2011, Remy et al. 2012, Wang et al. 2016). Indeed, PHT1 family genes including PHT1;1, PHT1;4, PHT1;8 and PHT1;9 were down-regulated in the vpt1 single mutant under Pi-sufficient conditions, which could be attributed to an elevated cytosol Pi level in the vpt1 mutant. In support of this idea, low Pi conditions failed to alter the expression of these PHT1 genes in vpt1 mutants (Fig. 4; Supplementary Table S2). Thus, less arsenic accumulation and enhanced As(V) tolerance in the vpt1 mutant could result from reduced expression of PHT1s due to altered intracellular Pi homeostasis (Fig. 6). Although molecular mechanisms of arsenate tolerance in plants are still not clear, our study on VPT1 provides a new perspective on the relationship between intracellular Pi balance and As(V) toxicity. Fig. 6 View largeDownload slide VPT1 is involved in arsenate resistance by modulating phosphate balance in Arabidopsis. Under As(V) toxicity conditions, both As(V) and Pi could be transported into cells through PHT1-type Pi transporters. The absorbed As(V) in cells is transformed into As(III) by arsenate reductase (AR), and then chelated by PCs, and subsequently loaded into the vacuole through ABCC1/2 phytochelatin transporters for arsenite detoxification. Meanwhile, the intracellular Pi homeostasis could affect the tolerance to As(V) in Arabidopsis. When VPT1 is knocked out, the intracellular Pi balance is altered, with more Pi arrested in the cytosol under Pi-sufficient conditions, then the expression of PHT1 family genes is down-regulated to restrict As(V) uptake. Thus VPT1-related intracellular Pi homeostasis contributes to As(V) tolerance in the plant cell. [The font size of the ‘Pi’ and ‘As(V)’ in the figure reflects the concentration of the ions, and the thickness of the arrows reflects the rate of ion flux.] Fig. 6 View largeDownload slide VPT1 is involved in arsenate resistance by modulating phosphate balance in Arabidopsis. Under As(V) toxicity conditions, both As(V) and Pi could be transported into cells through PHT1-type Pi transporters. The absorbed As(V) in cells is transformed into As(III) by arsenate reductase (AR), and then chelated by PCs, and subsequently loaded into the vacuole through ABCC1/2 phytochelatin transporters for arsenite detoxification. Meanwhile, the intracellular Pi homeostasis could affect the tolerance to As(V) in Arabidopsis. When VPT1 is knocked out, the intracellular Pi balance is altered, with more Pi arrested in the cytosol under Pi-sufficient conditions, then the expression of PHT1 family genes is down-regulated to restrict As(V) uptake. Thus VPT1-related intracellular Pi homeostasis contributes to As(V) tolerance in the plant cell. [The font size of the ‘Pi’ and ‘As(V)’ in the figure reflects the concentration of the ions, and the thickness of the arrows reflects the rate of ion flux.] Materials and Methods Plant materials and growth conditions Arabidopsis thaliana Col-0 seedlings were used as the wild type in the various experiments. The vpt1-1 (SAIL_96_H01) and vpt1-2 (SALK_006647) T-DNA insertion mutant lines were obtained from the Arabidopsis Biological Resource Center (ABRC). The homologous mutants were validated through PCR analysis. The primers are listed in Supplementary Table S1. Seeds were surface sterilized by 75% ethanol and sown on 1/2 MS agar plates (pH 5.8) with 1% sucrose, which is the control culture medium in this study. The plates with seeds were placed at 4°C for 2 d for vernalization. Then all plates were transferred to culture chambers at 22°C under a 16 h light/8 h dark photoperiod. The sufficient or control Pi medium is referred to as 1/2 MS medium (0.625 mM Pi), and the deficient Pi medium is 1/2 MS medium with 20 µM Pi. For arsenic toxicity treatment, NaH2AsO4 and NaAsO2 are used for As(V) and As(III) resistance assays. Quantitative PCR analysis The wild-type seedlings under sufficient and deficient Pi concentrations in the medium were harvested. Trizol (Invitrogen) solution was used to extract total RNA and then the RNA was treated with DNase I (Ambion) for genomic DNA depletion. A 2–3 µg aliquot of RNA was used for cDNA synthesis (Promega M-MLV Reverse Transcriptase). qPCR was performed on the Bio-rad CFX connect real-time system by using Fast start Universal SYBR Green Master Mix (Roche). The running protocol is 95°C for 10 min, 95°C for 15 s and 60°C for 1 min, 40 cycles; melt curve 65–95°C, 0.5°C for 0.05 s. The primers used for qPCR are listed in Supplementary Table S1. Determination of anthocyanin contents Leaves of the wild type and the vpt1 mutant were obtained for anthocyanin content measurements by using the method described by Teng et al. (2005). A 50 mg aliquot of each sample was washed in distilled water and then ground with liquid nitrogen. The powder samples were transferred in 2.5 ml of MeOH solution with 1% HCl (v/v) and stored at 4°C in the dark for 1 d. After centrifugation, the supernatant was used for anthocyanin content measurements. One anthocyanin unit is represented by one absorbance unit (A530–0.25A657) in 1 ml of extraction solution. Measurements of the content of Pi Wild-type Arabidopsis seedlings and VPT1-overexpressing lines were collected for Pi determination. The seedlings were washed three times in distilled water. A 50 mg aliquot of plant materials was gathered for Pi content detection with the ascorbate–molybdate–antimony method (John 1970). The samples were ground to a homogenate with distilled water (0.1 g FW/1 ml of H2O). After centrifugation, the supernatant was used for Pi measurements. Measurements of the content of arsenic For total arsenic analysis, 8-day-old seedlings were transferred onto 1/2 MS agar medium with 500 μM As(V) and grown vertically for 80 h. The seedlings were sampled at 0, 10, 20, 40 and 80 h for arsenic content assays. Each sample contains approximately 50 plants which were washed three times in a wash buffer [(1 mM K2HPO4, 0.5 mM Ca(NO3)2 and 5 mM MES (pH 6.0)]. Then the samples were kept at 80°C for 2 d, followed by digestion with 0.5 ml of reagent grade HNO3. After HNO3 treatment, the digested liquid was diluted with 10 ml of distilled water. Arsenic contents were analyzed with an ICP atomic emission spectrometer (NexION 300 ICP-MS). Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by National Natural Science Foundation of China Grants grant number 31670236 (to S. L.) and 31770267 (to W.-Z. L.) Acknowledgments We are grateful for the valuable advice on the As(V) sensitivity assay from Dr. Yanshan Chen (School of Environment, Nanjing University). Disclosures The authors have no conflicts of interest to declare. References Bleeker P.M. , Hakvoort H.W. , Bliek M. , Souer E. , Schat H. ( 2006 ) Enhanced arsenate reduction by a CDC25-like tyrosine phosphatase explains increased phytochelatin accumulation in arsenate-tolerant Holcus lanatus . Plant J . 45 : 917 – 929 . Bundschuh J. , Nath B. , Bhattacharya P. , Liu C.W. , Armienta M.A. , Moreno Lopez M.V. , et al. 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( 2015 ) Anaerobic arsenite oxidation by an autotrophic arsenite-oxidizing bacterium from an arsenic-contaminated paddy soil . Environ. Sci. Technol . 49 : 5956 – 5964 . Abbreviations Abbreviations ABCC1/2 C-type ATP-binding cassette transporter 1 and 2 AR arsenate reductase As(III) arsenite As(V) arsenate ER endoplasmic reticulum GUS β-glucuronidase MS Murashige and Skoog NIP nodulin 26-like intrinsic proteins PC phytochelatin PHT1 phosphate transporter 1 Pi phosphate qPCR quantitative real-time PCR VPT1 vacuolar phosphate transporter 1 © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

Vacuolar Phosphate Transporter 1 (VPT1) Affects Arsenate Tolerance by Regulating Phosphate Homeostasis in Arabidopsis

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Abstract

Abstract Arsenate [As(V)] is toxic to nearly all organisms. Soil-borne As(V) enters plant cells mainly through the plasma membrane-localized phosphate (Pi) transporter PHT1 family proteins due to its chemical similarity to Pi. We report here that VPT1, a major vacuolar phosphate transporter which contributes to vacuolar Pi sequestration, is associated with As(V) tolerance in Arabidopsis. vpt1 mutants displayed enhanced tolerance to As(V) toxicity, whereas plants overexpressing VPT1 were more sensitive to As(V) as compared with the wild-type plants. Measurements of arsenic content indicated that vpt1 mutants accumulated less arsenic and, in contrast, up-regulating VPT1 expression contributed to higher levels of arsenic accumulation in plants. To examine further how VPT1 may modulate arsenic contents in plants, we surveyed the expression patterns of all the PHT1 family members that play roles in As(V) uptake, and found that many of the PHT1 genes were down-regulated in the vpt1 mutant as compared with the wild type under Pi-sufficient conditions, but not when Pi levels were low in the medium. Interestingly, As(V) sensitivity assays indicated that As(V) resistance in vpt1 mutants was prominent only under Pi-sufficient but not under Pi-deficient conditions. These results suggest that under Pi-sufficient conditions, loss of VPT1 leads to elevated levels of Pi in the cytosol, which in turn suppressed the expression of PHT1-type transporters and reduced accumulation of arsenic. Introduction Oxidized arsenic species are identified as a non-threshold class-I human carcinogen (Salt 2017). Arsenic contamination has become increasingly severe in soil and water resources due to industrial wastewater discharge and application of pesticides in agriculture (Moreno-Jimenez et al. 2012). Arsenic exposure through drinking water and food negatively impacts human health, leading to increased risk of cancer and other diseases (Bundschuh et al. 2012, Joseph et al. 2015). Soil-borne arsenic can accumulate in the plant root system and inhibit cell division and elongation (Garg and Singla 2011). When transported to the upper parts of a plant, arsenic damages the chloroplast structure and causes a decline in plant growth (Finnegan and Chen 2012). Arsenic exists in two inorganic forms, namely arsenate [As(V)] and arsenite [As(III)]. As(V) is a chemical analog of phosphate (Pi), and may disrupt Pi-dependent aspects of metabolism (Raghothama 1999, Tu and Ma 2003, Wu et al. 2011). As(III) binds to the sulfhydryl group of cysteine in many proteins with a high affinity, and then severely affects protein structure, leading to adverse effects on many metabolic processes (Kitchin and Wallace 2006). Arsenic in the soil is absorbed by plants and may accumulate in edible parts such as fruits and seeds (Williams et al. 2007, Sohn 2014). Therefore, understanding the arsenic transport mechanisms in plants is critical to development of genetic tools that limit arsenic accumulation in crop species. As(III) mainly exists in anaerobic soil such as paddy fields which are used for cultivating rice (Xu et al. 2008). Studies indicate that As(III) is transported into plants mainly through NIP (nodulin 26-like intrinsic proteins) family proteins (Ma et al. 2008, Xu et al. 2015). Disruption of NIP1;1 function significantly enhances the resistance to arsenite in Arabidopsis (Kamiya et al. 2009). NIP1;3, another NIP family member, is responsible for root to shoot translocation of As(III) (Xu et al. 2015). When As(III) is transported into cells, it can be chelated by phytochelatins (PCs) as As(III)–PCs, and then this complex is sequestrated by the vacuole through ABCC1/2 (C-type ABC transporters 1 and 2) (Song et al. 2010). As(V) is a primary arsenic form in aerobic soils (Moreno-Jimenez et al. 2012), and accounts for 5–20% of total soluble arsenic in anaerobic soil (Khan et al. 2010, Zhang et al. 2015). Due to the chemical similarity to Pi, As(V) enters plant cells mainly through the high-affinity Pi transport systems, especially PHT1 (phosphate transporter1)-type transporters (Shin et al. 2004, Catarecha et al. 2007). Thus, mutations of genes encoding these transporters may reduce As(V) uptake and sensitivity to As(V) stress in plants. For instance, loss of function of PHT1;1 and PHT1;4, the major Pi transporters involved in Pi uptake, confers enhanced As(V) tolerance in Arabidopsis (Shin et al. 2004). Loss of function of PHT1;9, another plasma membrane-localized phosphate transporter, reduces arsenic content in plants under As(V) toxic conditions, while overexpressing PHT1;9 results in enhanced arsenic accumulation (Remy et al. 2012). Similarly, the knockout of OsPT8, a plasma membrane-localized PHT family member in rice, reduces the As(V) content to about 50% of that of control plants (Wang et al. 2016), whereas its overexpression causes overaccumulation of arsenic in plants (Wu et al. 2011). Moreover, regulatory proteins governing the expression of PHT1-type genes are also shown to play a role in As(V) tolerance. The WRKY6 transcription factor restricts arsenate uptake efficiency by inhibiting the expression of PHT1;1 (Castrillo et al. 2013). Loss of phosphate transporter traffic facilitator1 (PHF1), an endoplasmic reticuulum (ER) protein that facilitates PHT1;1 targeting to the plasma membrane, also enhances As(V) tolerance (Gonzalez et al. 2005). In contrast, up-regulation of OsPHR2, a MYB transcriptional factor gene that activates the expression of OsPT genes, would contribute to As(V) uptake (Wu et al. 2011). Therefore, plasma membrane-localized PHT1-type Pi transporters are closely related to arsenic accumulation in plants. Furthermore, Pi status in the soil and in plants also contributes to the extent of As(V) toxicity. It has been shown that low Pi conditions exacerbate plant sensitivity to As(V), while Pi application to the soil enhances As(V) tolerance in plants (Tu and Ma 2003, Catarecha et al. 2007). Taken together, a better understanding of the cross-talk between Pi balance and As(V) tolerance should contribute to the knowledge base for improving As(V) tolerance in plants. Studies so far have been focused on the relevance of plasma membrane-localized Pi transporters (PHT1s) for their role in As(V) uptake as described above. However, the endomembrane-localized Pi transporters and their contribution to As(V) toxicity have not been addressed. In particular, the vacuole works as the major pool of inorganic phosphorus in the plant cells and plays a key role for Pi homeostasis maintaining in plants (Pratt et al. 2009). Recent findings showing that vacuolar phosphate transporter 1 (VPT1; or PHT5; 1) as the major Pi transporter responsible for vacuolar Pi accumulation (Liu et al. 2015, T.Y. Liu et al. 2016), providing an opportunity for examining its potential role in As(V) toxicity. In this study, we found that vpt1 mutants displayed enhanced tolerance to As(V) toxicity specifically under Pi-sufficient conditions possibly by inhibiting the expression of PHT1-type transporters. Results and Discussion vpt1 mutants display enhanced As(V) tolerance Phosphate transporters (such as PHT1 family proteins) are critical in fine-tuning Pi homeostasis in plant cells, which affect the sensitivity to As(V) stress (Li et al. 2016). VPT1 has recently been identified as a major vacuolar phosphate transporter, and disruption of its function alters Pi homeostasis, with more Pi arrested in the cytosol (Liu et al. 2015, J. Liu et al. 2016). As Pi is closely related to As(V) tolerance in plants, we expected that VPT1 may affect As(V) tolerance in plants. To test this hypothesis, we compared two Arabidopsis mutant lines containing T-DNA insertional alleles of the VPT1 gene (vpt1-1 and vpt1-2) with wild-type plants under various concentrations of As(V) in the culture medium. After germination in normal half-strength Murashige and Skoog (1/2 MS) medium for 4 d, the plants were transferred to medium containing 0, 250, 500 or 700 µM As(V). As shown in Fig. 1A, the growth of all seedlings was severely inhibited by increasing As(V) concentrations. However, vpt1 mutant plants consistently displayed more tolerance to these As(V) treatments as compared with the wild-type plants (Fig. 1B). For instance, the fresh weight of the vpt1 mutant was significantly higher than that of the wild type once the As(V) concentration reached 250 µM and beyond. At 700 µM, As(V) severely inhibited the growth of wild-type seedlings that displayed a darker color, reflecting elevated levels of anthocyanin [Fig. 1A;Supplementary Fig. S1]. In addition to larger cotyledons (Supplementary Fig. S1), vpt1 mutants also had longer roots than the wild type (Fig. 1C). The length of vpt1 roots was nearly 50% longer than that of the wild-type plants when grown on the culture medium with 700 µM As(V). Fig. 1 View largeDownload slide vpt1 single mutants are less sensitive to toxic levels of As(V). (A) Wild-type and vpt1 seedlings under different As(V) conditions. Four-day old seedlings from a 1/2 MS plate were transferred onto plates with 0 µM (I), 250 µM (II), 500 µM (III) and 700 µM (IV, V) As(V) and cultured for 7 d. Scale bars represent 1 cm (I, II, III and IV) and 1 mm (V). The single seedling fresh weight and primary root length (B, C) were measured for phenotyping. The results were reproducible in three independent experiments. Data are the mean ± SD. n = 8. Asterisks represent a significant difference between vpt1 mutant lines and the wild type under the same culture conditions. Student’s t-test, *P < 0.05, **P < 0.01. Fig. 1 View largeDownload slide vpt1 single mutants are less sensitive to toxic levels of As(V). (A) Wild-type and vpt1 seedlings under different As(V) conditions. Four-day old seedlings from a 1/2 MS plate were transferred onto plates with 0 µM (I), 250 µM (II), 500 µM (III) and 700 µM (IV, V) As(V) and cultured for 7 d. Scale bars represent 1 cm (I, II, III and IV) and 1 mm (V). The single seedling fresh weight and primary root length (B, C) were measured for phenotyping. The results were reproducible in three independent experiments. Data are the mean ± SD. n = 8. Asterisks represent a significant difference between vpt1 mutant lines and the wild type under the same culture conditions. Student’s t-test, *P < 0.05, **P < 0.01. Arsenic is present mainly as As(V) and As(III) that are both toxic to plants. Several studies suggested that As(V) in the cytosol could be rapidly reduced to As(III) by arsenate reductase (AR) (Bleeker et al. 2006, Duan et al. 2007, Salt 2017). In fact, As(III) is much more toxic to plant cells than As(V). Studies indicated that micromolar levels of As(III) can be inhibitory to plant growth (Ji et al. 2017). To test if enhanced As(V) tolerance in vpt1 was a result of altered sensitivity to As(III) derived from As(V), we cultured the seedlings on 1/2 MS medium with various concentrations of As(III). The experimental results indicated that As(III) was indeed more toxic to plants and 5 µM already severely inhibited the growth of Arabidopsis plants (Fig. 2A). However, the vpt1 mutant and the wild-type plants showed similar sensitivity to As(III), as measured by root length and fresh weight (Fig. 2B, C). Additionally, we surveyed the expression patterns of genes involved in As(III) resistance, including ACR2, ABCC1, ABCC2 and NIP1;1 (Ma et al. 2008, Kamiya et al. 2009, Song et al. 2010). The quantitative real-time PCR (qPCR) data indicated that the expression levels of these genes were similar in vpt1 and wild-type plants (Supplementary Fig. S2). This suggests that loss of function of VPT1 specifically affects the sensitivity to As(V) in Arabidopsis. Fig. 2 View largeDownload slide VPT1s are not related to As(III) resistance. (A) Various genotypes showed no As(III) resistance. Four-day old seedlings of various genotypes from a 1/2 MS plate were transferred on plates with 2, 5 and 9 µM As(III) separately and cultured for 7 d. The fresh weight and root length are presented in (B) and (C). Data are the mean ± SD, n = 4. The results were reproducible in three independent experiments; scale bars indicate 2 cm. Fig. 2 View largeDownload slide VPT1s are not related to As(III) resistance. (A) Various genotypes showed no As(III) resistance. Four-day old seedlings of various genotypes from a 1/2 MS plate were transferred on plates with 2, 5 and 9 µM As(III) separately and cultured for 7 d. The fresh weight and root length are presented in (B) and (C). Data are the mean ± SD, n = 4. The results were reproducible in three independent experiments; scale bars indicate 2 cm. VPT1 overexpression enhances arsenate sensitivity in Arabidopsis To examine further the function of VPT1 in plant As(V) stress, we overexpressed VPT1 in wild-type Arabidopsis. The Pi content measurements showed that overexpressing VPT1 leads to increased Pi accumulation in Arabidopsis (Supplementary Fig. S3), which is also indicated by other studies (Liu et al. 2015, T.Y. Liu et al. 2016). We selected two transgenic lines (OV1-1 and OV1-2) with higher levels of VPT1 transcripts for phenotyping. The As(V) sensitivity assays showed that OV1-1 and OV1-2 seedlings were more sensitive to As(V) stress as compared with the wild-type seedlings, while the vpt1 single mutant displayed enhanced tolerance to As(V) as described earlier (Fig. 3A). For instance, 400 µM As(V) inhibited root growth and reduced the fresh weight of the seedlings in all genotypes, but OV1 plants were more affected than the wild type and the vpt1 mutant (Fig. 3B, C). There appeared to be a positive correlation between VPT1 expression level and As(V) sensitivity (Supplementary Figs. S3, S4). For instance, OV1-1 and OV1-2 seedlings showed higher levels of VPT1 mRNA (about 21 times higher than the wild type), correlated with shorter roots and lower fresh weight under As(V) stress, whereas OV1-3 seedlings had a lower VPT1 mRNA level (about six times higher than the wild type) as compared with OV1-1/OV1-2 and were less sensitive to As(V) toxicity (Supplementary Figs. S3, S4). The OV1-4 seedling exhibited the least sensitivity to As(V) among the VPT1-overexpressing lines and its VPT1 expression level was about twice as high as that in wild-type seedlings. Fig. 3 View largeDownload slide VPT1 modulates sensitivity to arsenate in Arabidopsis. (A) Phenotypes of various genotypes under As(V) toxic conditions. Three-day-old seedlings from a 1/2 MS plate were transferred to a plate with 400 µM As(V) and cultured for 6 d. Scale bars represent 1 cm. Primary root length (B) and the single seedling fresh weight (C) were measured for phenotyping. The results were reproducible in three independent experiments. Data are the mean ± SD, n = 8. Different letters indicate a significant difference among various seedling lines under the same culture conditions. Student’s t-test, **P < 0.01. (D) VPT1 is related to As(V) accumulation in Arabidopsis. Different 8-day-old genotypes were transferred to medium with 400 µM As(V) and sampled at 0, 1, 2, 3 and 4 d for arsenic measurements. Data are the mean ± SD, n = 4. Fig. 3 View largeDownload slide VPT1 modulates sensitivity to arsenate in Arabidopsis. (A) Phenotypes of various genotypes under As(V) toxic conditions. Three-day-old seedlings from a 1/2 MS plate were transferred to a plate with 400 µM As(V) and cultured for 6 d. Scale bars represent 1 cm. Primary root length (B) and the single seedling fresh weight (C) were measured for phenotyping. The results were reproducible in three independent experiments. Data are the mean ± SD, n = 8. Different letters indicate a significant difference among various seedling lines under the same culture conditions. Student’s t-test, **P < 0.01. (D) VPT1 is related to As(V) accumulation in Arabidopsis. Different 8-day-old genotypes were transferred to medium with 400 µM As(V) and sampled at 0, 1, 2, 3 and 4 d for arsenic measurements. Data are the mean ± SD, n = 4. A recent study indicates that As(V) inhibits root hair growth (Forino et al. 2012). We thus examined the effect of As(V) on root hair growth in plants of different genotypes (Supplementary Fig. S5). Under control condition without As(V), all plants had similar root hairs of 200–600 µm long (Supplementary Fig. S5A, B). In the presence of 400 µM As(V) in the medium, the length of root hairs was in the range of 50–200 µm in OV1 plants, 100–300 µm in wild-type seedlings and 200–500 µm in the vpt1 mutant plants (Supplementary Fig. S5A, C). This supports the notion that overexpression of VPT1 causes hypersensitivity to As(V) in plants. Given that As(V) uptake directly affects the sensitivity to As(V) toxicity in plants (Shin et al. 2004, Wu et al. 2011), we suspected that the amount of As(V) absorbed by plants of different genotypes (the wild type, vpt1 mutant and VPT1-overexpressing lines) may be different and would directly correlate with inhibition of plant growth (Shin et al. 2004, Wu et al. 2011). After 8-day-old seedlings were transferred to medium with 400 µM As(V) and cultured for a further 4 d, we measured arsenic contents of different plants and found that vpt1-1 mutant plants contained less arsenic while OV1-1 seedlings accumulated more arsenic, as compared with the wild type (Fig. 3D). On day 4 after As(V) treatment, the arsenic content of the vpt1-1 mutant was about 23% less than in the wild type, while OV1-1 plants contained 35% more arsenic than wild-type seedlings. This is consistent with the extent of toxicity observed in these plants. VPT1-associated As(V) sensitivity depends on Pi status In general, inhibition of As(V) uptake will decrease arsenic accumulation in plants (Remy et al. 2012, Wang et al. 2016). As(V) could be transported by Pi transporters due to its chemical similarity to phosphate, and plasma membrane-localized Pi transporters are known to be responsible for arsenate acquisition. However, VPT1 is a vacuolar phosphate transporter (Liu et al. 2015, T.Y. Liu et al. 2016) and it is not known how VPT1 may affect arsenic accumulation in Arabidopsis. In particular, As(V) is largely converted into As(III) in the cytosol and we do not expect a role for VPT1 in vacuolar sequestration of As(V) (Bleeker et al. 2006, Duan et al. 2007, Salt 2017). Our previous study showed that loss of VPT1 leads to higher levels of Pi in the cytosol (Liu et al. 2015), leading to down-regulation of PHT1;1 and PHT1;4 under Pi-sufficient conditions. A number of previous studies have shown that reduced expression levels of PHT1 genes would enhance As(V) tolerance in plants (Shin et al. 2004, Catarecha et al. 2007, Remy et al. 2012, Wang et al. 2016). Therefore, lower expression levels of PHT1 family members may account for reduced uptake of As(V) in the vpt1 mutant. We analyzed the expression patterns of PHT1 family members in the vpt1 mutant and wild-type plants, and found that many of the PHT1 family genes, including PHT1;1, PHT1;2, PHT1;3, PHT1;4, PHT1;8 and PHT1;9, were significantly down-regulated in the vpt1 mutant as compared with wild-type plants under a Pi-sufficient condition (0.625 mM) (Fig. 4A). Furthermore, we analyzed the expression levels of the PHT1 gene in the vpt1 mutant grown under various Pi conditions (i.e. 20, 50, 200 and 625 µM, and 2 mM). We found that PHT1 genes in the vpt1 mutant were also induced as the Pi concentration in the medium decreased (Supplementary Table S2). When the seedlings grew under a Pi-deficient condition (20 µM Pi), the expression levels of PHT1 genes (i.e. PHT1;1, PHT1;3, PHT1;4, PHT1;7, PHT1;8 and PHT1;9) in the vpt1 mutant were comparable with those in the wild type (Fig. 4B). Fig. 4 View largeDownload slide The expression of PHT1 family genes is altered in the vpt1 mutant. (A and B) The expression patterns of PHT1 family members in the wild type and the vpt1 single mutant. Ten-day-old seedlings that were cultured in Pi-sufficient (0.625 mM) and Pi-deficient (20 µM) medium were obtained for determination of the PHT1 gene expression level. Wild-type seedlings were used as control; values were normalized to UBQ10. Data are the mean ± SD, n = 4. Statistical analysis was conducted between wild-type (Col-0) and vpt1 mutant plants by using Student’s t-test (*P < 0.05, **P < 0.01). Fig. 4 View largeDownload slide The expression of PHT1 family genes is altered in the vpt1 mutant. (A and B) The expression patterns of PHT1 family members in the wild type and the vpt1 single mutant. Ten-day-old seedlings that were cultured in Pi-sufficient (0.625 mM) and Pi-deficient (20 µM) medium were obtained for determination of the PHT1 gene expression level. Wild-type seedlings were used as control; values were normalized to UBQ10. Data are the mean ± SD, n = 4. Statistical analysis was conducted between wild-type (Col-0) and vpt1 mutant plants by using Student’s t-test (*P < 0.05, **P < 0.01). In parallel, we performed an As(V) tolerance assay under various Pi levels (Fig. 5A). On the culture medium with a low level of Pi (20 µM), the vpt1 mutant and the wild-type plants were similarly sensitive to As(V). As the Pi concentration was elevated, vpt1 became less sensitive to As(V) stress as compared with the wild type. When the Pi concentration reached 0.625 mM (sufficient Pi condition), the root length and fresh weight of the vpt1 mutant were about 31% and 45% more than in wild-type plants (Fig. 5B–E). However, under high Pi conditions (2 mM), the sensitivities to As(V) in vpt1 and the wild type were difficult to assess because vpt1 is more sensitive to high Pi conditions (Fig. 5A, E) (Liu et al. 2015). Moreover, arsenic content in the vpt1 mutant was lower than in the wild type when the Pi concentration in the medium was >200 µM, whereas such a difference disappeared under lower Pi conditions (<50 µM) (Supplementary Fig. S6). Thus, the sensitivity to As(V) stress in the vpt1 mutant depends on the Pi status, and altered cytosol Pi homeostasis might be related to the enhanced As(V) tolerance in the vpt1 mutant. Fig. 5 View largeDownload slide The vpt1-related resistance to As(V) depends on Pi status. (A) As(V) sensitivity assays under various Pi conditions. Three-day old seedlings were transferred to 1/2 MS medium plates with various concentrations of Pi (i.e. 20, 50, 200 and 625 µM, and 2 mM) and cultured for 7 d. As(V) at 400 µM was applied for As(V) sensitivity assays; scale bars represent 2 cm. The root length (B, C) and fresh weight (D, E) were used for qualification of As(V) sensitivity assays results. Data are the mean ±SD, n = 8. Student’s t-test (*P < 0.05, **P < 0.01). Fig. 5 View largeDownload slide The vpt1-related resistance to As(V) depends on Pi status. (A) As(V) sensitivity assays under various Pi conditions. Three-day old seedlings were transferred to 1/2 MS medium plates with various concentrations of Pi (i.e. 20, 50, 200 and 625 µM, and 2 mM) and cultured for 7 d. As(V) at 400 µM was applied for As(V) sensitivity assays; scale bars represent 2 cm. The root length (B, C) and fresh weight (D, E) were used for qualification of As(V) sensitivity assays results. Data are the mean ±SD, n = 8. Student’s t-test (*P < 0.05, **P < 0.01). In summary, the above results have connected Pi homeostasis and As(V) toxicity through the study of VPT1, a tonoplast-localized phosphate transporter responsible for vacuolar Pi sequestration (Liu et al. 2015, T.Y. Liu et al. 2016). The vacuole is the major phosphate storage space in plant cells, and its Pi concentration often reaches >10 mM, whereas for the cytosol, its Pi concentration is only about 60 μM (Pratt et al. 2009). When Pi is at sufficient or excess levels, the vacuole sequesters extra Pi that becomes remobilized under Pi-deficient conditions. Therefore, VPT1 is critical for Pi homeostasis in plants for its role in vacuolar Pi accumulation. As a result, the vpt1 mutant is less capable of adapting to changing Pi status in the environment. Loss of VPT1 would also severely reduce vacuolar Pi content and cause elevated Pi levels in the cytosol. The altered intracellular Pi homeostasis in vpt1 mutant cells contributes to altered expression of PSR (phosphate starvation response) genes (Liu et al. 2015, T.Y. Liu et al. 2016), including those encoding PHT1 family Pi uptake transporters which are also responsible for As(V) uptake (Shin et al. 2004, Catarecha et al. 2007, Wu et al. 2011, Remy et al. 2012, Wang et al. 2016). Indeed, PHT1 family genes including PHT1;1, PHT1;4, PHT1;8 and PHT1;9 were down-regulated in the vpt1 single mutant under Pi-sufficient conditions, which could be attributed to an elevated cytosol Pi level in the vpt1 mutant. In support of this idea, low Pi conditions failed to alter the expression of these PHT1 genes in vpt1 mutants (Fig. 4; Supplementary Table S2). Thus, less arsenic accumulation and enhanced As(V) tolerance in the vpt1 mutant could result from reduced expression of PHT1s due to altered intracellular Pi homeostasis (Fig. 6). Although molecular mechanisms of arsenate tolerance in plants are still not clear, our study on VPT1 provides a new perspective on the relationship between intracellular Pi balance and As(V) toxicity. Fig. 6 View largeDownload slide VPT1 is involved in arsenate resistance by modulating phosphate balance in Arabidopsis. Under As(V) toxicity conditions, both As(V) and Pi could be transported into cells through PHT1-type Pi transporters. The absorbed As(V) in cells is transformed into As(III) by arsenate reductase (AR), and then chelated by PCs, and subsequently loaded into the vacuole through ABCC1/2 phytochelatin transporters for arsenite detoxification. Meanwhile, the intracellular Pi homeostasis could affect the tolerance to As(V) in Arabidopsis. When VPT1 is knocked out, the intracellular Pi balance is altered, with more Pi arrested in the cytosol under Pi-sufficient conditions, then the expression of PHT1 family genes is down-regulated to restrict As(V) uptake. Thus VPT1-related intracellular Pi homeostasis contributes to As(V) tolerance in the plant cell. [The font size of the ‘Pi’ and ‘As(V)’ in the figure reflects the concentration of the ions, and the thickness of the arrows reflects the rate of ion flux.] Fig. 6 View largeDownload slide VPT1 is involved in arsenate resistance by modulating phosphate balance in Arabidopsis. Under As(V) toxicity conditions, both As(V) and Pi could be transported into cells through PHT1-type Pi transporters. The absorbed As(V) in cells is transformed into As(III) by arsenate reductase (AR), and then chelated by PCs, and subsequently loaded into the vacuole through ABCC1/2 phytochelatin transporters for arsenite detoxification. Meanwhile, the intracellular Pi homeostasis could affect the tolerance to As(V) in Arabidopsis. When VPT1 is knocked out, the intracellular Pi balance is altered, with more Pi arrested in the cytosol under Pi-sufficient conditions, then the expression of PHT1 family genes is down-regulated to restrict As(V) uptake. Thus VPT1-related intracellular Pi homeostasis contributes to As(V) tolerance in the plant cell. [The font size of the ‘Pi’ and ‘As(V)’ in the figure reflects the concentration of the ions, and the thickness of the arrows reflects the rate of ion flux.] Materials and Methods Plant materials and growth conditions Arabidopsis thaliana Col-0 seedlings were used as the wild type in the various experiments. The vpt1-1 (SAIL_96_H01) and vpt1-2 (SALK_006647) T-DNA insertion mutant lines were obtained from the Arabidopsis Biological Resource Center (ABRC). The homologous mutants were validated through PCR analysis. The primers are listed in Supplementary Table S1. Seeds were surface sterilized by 75% ethanol and sown on 1/2 MS agar plates (pH 5.8) with 1% sucrose, which is the control culture medium in this study. The plates with seeds were placed at 4°C for 2 d for vernalization. Then all plates were transferred to culture chambers at 22°C under a 16 h light/8 h dark photoperiod. The sufficient or control Pi medium is referred to as 1/2 MS medium (0.625 mM Pi), and the deficient Pi medium is 1/2 MS medium with 20 µM Pi. For arsenic toxicity treatment, NaH2AsO4 and NaAsO2 are used for As(V) and As(III) resistance assays. Quantitative PCR analysis The wild-type seedlings under sufficient and deficient Pi concentrations in the medium were harvested. Trizol (Invitrogen) solution was used to extract total RNA and then the RNA was treated with DNase I (Ambion) for genomic DNA depletion. A 2–3 µg aliquot of RNA was used for cDNA synthesis (Promega M-MLV Reverse Transcriptase). qPCR was performed on the Bio-rad CFX connect real-time system by using Fast start Universal SYBR Green Master Mix (Roche). The running protocol is 95°C for 10 min, 95°C for 15 s and 60°C for 1 min, 40 cycles; melt curve 65–95°C, 0.5°C for 0.05 s. The primers used for qPCR are listed in Supplementary Table S1. Determination of anthocyanin contents Leaves of the wild type and the vpt1 mutant were obtained for anthocyanin content measurements by using the method described by Teng et al. (2005). A 50 mg aliquot of each sample was washed in distilled water and then ground with liquid nitrogen. The powder samples were transferred in 2.5 ml of MeOH solution with 1% HCl (v/v) and stored at 4°C in the dark for 1 d. After centrifugation, the supernatant was used for anthocyanin content measurements. One anthocyanin unit is represented by one absorbance unit (A530–0.25A657) in 1 ml of extraction solution. Measurements of the content of Pi Wild-type Arabidopsis seedlings and VPT1-overexpressing lines were collected for Pi determination. The seedlings were washed three times in distilled water. A 50 mg aliquot of plant materials was gathered for Pi content detection with the ascorbate–molybdate–antimony method (John 1970). The samples were ground to a homogenate with distilled water (0.1 g FW/1 ml of H2O). After centrifugation, the supernatant was used for Pi measurements. Measurements of the content of arsenic For total arsenic analysis, 8-day-old seedlings were transferred onto 1/2 MS agar medium with 500 μM As(V) and grown vertically for 80 h. The seedlings were sampled at 0, 10, 20, 40 and 80 h for arsenic content assays. Each sample contains approximately 50 plants which were washed three times in a wash buffer [(1 mM K2HPO4, 0.5 mM Ca(NO3)2 and 5 mM MES (pH 6.0)]. Then the samples were kept at 80°C for 2 d, followed by digestion with 0.5 ml of reagent grade HNO3. After HNO3 treatment, the digested liquid was diluted with 10 ml of distilled water. Arsenic contents were analyzed with an ICP atomic emission spectrometer (NexION 300 ICP-MS). Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by National Natural Science Foundation of China Grants grant number 31670236 (to S. L.) and 31770267 (to W.-Z. L.) Acknowledgments We are grateful for the valuable advice on the As(V) sensitivity assay from Dr. Yanshan Chen (School of Environment, Nanjing University). Disclosures The authors have no conflicts of interest to declare. References Bleeker P.M. , Hakvoort H.W. , Bliek M. , Souer E. , Schat H. ( 2006 ) Enhanced arsenate reduction by a CDC25-like tyrosine phosphatase explains increased phytochelatin accumulation in arsenate-tolerant Holcus lanatus . Plant J . 45 : 917 – 929 . Bundschuh J. , Nath B. , Bhattacharya P. , Liu C.W. , Armienta M.A. , Moreno Lopez M.V. , et al. 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( 2015 ) Anaerobic arsenite oxidation by an autotrophic arsenite-oxidizing bacterium from an arsenic-contaminated paddy soil . Environ. Sci. Technol . 49 : 5956 – 5964 . Abbreviations Abbreviations ABCC1/2 C-type ATP-binding cassette transporter 1 and 2 AR arsenate reductase As(III) arsenite As(V) arsenate ER endoplasmic reticulum GUS β-glucuronidase MS Murashige and Skoog NIP nodulin 26-like intrinsic proteins PC phytochelatin PHT1 phosphate transporter 1 Pi phosphate qPCR quantitative real-time PCR VPT1 vacuolar phosphate transporter 1 © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Plant and Cell PhysiologyOxford University Press

Published: Feb 6, 2018

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