Identification of C-terminal Regions in Arabidopsis thaliana Phytochelatin Synthase 1 Specifically Involved in Activation by Arsenite

Identification of C-terminal Regions in Arabidopsis thaliana Phytochelatin Synthase 1... Abstract Phytochelatins (PCs) are major chelators of toxic elements including inorganic arsenic (As) in plant cells. Their synthesis confers tolerance and influences within-plant mobility. Previous studies had shown that various metal/metalloid ions differentially activate PC synthesis. Here we identified C-terminal parts involved in arsenite- [As(III)] dependent activation of AtPCS1, the primary Arabidopsis PC synthase. The T-DNA insertion in the AtPCS1 mutant cad1-6 causes a truncation in the C-terminal regulatory domain that differentially affects activation by cadmium (Cd) and zinc (Zn). Comparisons of cad1-6 with the AtPCS1 null mutant cad1-3 and the double mutant of tonoplast PC transporters abcc1/2 revealed As(III) hypersensitivity of cad1-6 equal to that of cad1-3. Both cad1-6 and cad1-3 showed increased As distribution to shoots compared with Col-0, whereas Zn accumulation in shoots was equally lower in cad1-6 and cad1-3. Supporting these phenotypes of cad1-6, PC accumulation in the As(III)-exposed plants were at trace level in both cad1-6 and cad1-3, suggesting that the truncated AtPCS1 of cad1-6 is defective in PCS activity in response to As(III). Analysis of a C-terminal deletion series of AtPCS1 using the PCS-deficient mutant of fission yeast suggested important regions within the C-terminal domain for As(III)-dependent PC synthesis, which were different from the regions previously suggested for Cd- or Zn-dependent activation. Interestingly, we identified a truncated variant more strongly activated than the wild-type protein. This variant could potentially be used as a tool to better restrict As mobility in plants. Introduction Arsenic (As) is ranked first on the US Agency for Toxic Substances and Disease Registry (ATSDR) 2013 Priority List of Hazardous Substances (http://www.atsdr.cdc.gov/spl). Long-term exposure to low levels of As has been associated with various human diseases including cancer and cardiovascular disease, as well as higher overall mortality (Jomova et al. 2011). Thus As accumulation in plants harbors a potential risk to human health as a source of As intake (Meharg 2004, Gilbert-Diamond et al. 2011). Control over As accumulation in plants is needed to achieve mitigation of human As exposure through plant-derived food ingestion. Inorganic forms of As, namely arsenite [As(III)] and arsenate [As(V)], are more toxic to organisms than organic forms such as arsenosugar and arsenobetaine. Plants absorb both As(III) and As(V) through root-expressed members of the aquaporin (Ma et al. 2008, Kamiya et al. 2009, Xu et al. 2015) and phosphate transporter families , respectively (Shin et al. 2004, Wu et al. 2011, Kamiya et al. 2013). Absorbed As(V) is rapidly reduced to As(III) by arsenate reductases in root cells (Chao et al. 2014, Sánchez-Bermejo et al. 2014, Shi et al. 2016, Xu et al. 2017). Therefore plant cells are challenged mainly by As(III) after exposure to either As(V) or As(III). As(III) toxicity has been attributed to its high affinity for sulfhydryl groups. As(III) can bind to reduced cysteines in peptides and proteins such as zinc-finger proteins (Zhao et al. 2012, Shen et al. 2013). Glutathione (GSH), a major cysteine-containing peptide in cells, can form a complex with As(III) in a molar ratio of 3 : 1 (Scott et al. 1993, Spuches et al. 2005). In plant cells, phytochelatins (PCs) are synthesized by PC synthases (PCSs) from GSH when plants are exposed to non-essential toxic ions as well as essential metals such as zinc (Zn) and copper (Cu). PCs are polypeptides with the general structure (γ-Glu–Cys)n–Gly (usually n = 2–7, referred to as PC2–PC7) and are major chelators of toxic elements including inorganic As in plant cells. For As(III), it has been demonstrated in vitro that a PC2–As(III) complex is formed with three of four cysteines in two molecules of PC2 (Schmöger et al. 2000). Various PC–As species have been detected in plants (Liu et al. 2010) and the chelation of As(III) with PCs is crucial for As(III) detoxification in the plant cells. In Arabidopsis, AtPCS1 is the primary PCS synthesizing PCs in response to toxic inorganic ions of As, cadmium (Cd), mercury (Hg) and lead (Pb) (Howden et al. 1995, Ha et al. 1999, Fischer et al. 2014) as well as under conditions of Zn excess (Tennstedt et al. 2009). The AtPCS1 null mutant cad1-3 displays enhanced sensitivity to these toxic elements including As, suggesting a central role for AtPCS1 in toxic element detoxification. The PC–metal(loid) complexes are sequestered into the vacuoles by tonoplast PC transporters AtABCC1 and AtABCC2 (Song et al. 2010, Park et al. 2011). The rice ortholog OsABCC1 is correspondingly responsible for As–PC complex sequestration in vacuoles (Song et al. 2014). Thus, both PC synthesis and transport are crucial steps for achieving PC-dependent detoxification of toxic elements in plant cells. Another important role indicated for PC synthesis is its involvement in long-distance element transport within plants. For example, in rice, PC synthesis by OsPCS1 and 2 and vacuolar compartmentation of PC–As complexes mediated by OsABCC1 significantly retard long-distance As transport from root to shoot and further to grains (Song et al. 2014, Hayashi et al. 2017, Uraguchi et al. 2017). There are also some indications in Arabidopsis that AtPCS1-dependent PC synthesis controls long-distance transport of As as well as Cd and Zn (Chen et al. 2006, Liu et al. 2010, Kühnlenz et al. 2016). Despite these physiological functions of AtPCS1, especially in As detoxification and transport, mechanisms governing AtPCS1 activation by As remain to be elucidated. Higher plant PCSs have a general structure with two major domains (Rea 2012): the N-terminal catalytic domain (pfam05023) and the C-terminal Phytochelatin_C domain with unknown functions (pfam09328). In vitro experiments indicated that in addition to the N-terminal domain, different regions of the AtPCS1 C-terminal domain are crucial for metal-specific PCS activation (Ruotolo et al. 2004, Romanyuk et al. 2006). Recently it was also suggested that in planta Cd- and Zn-dependent PC synthesis require different regions of the AtPCS1 C-terminal domain (Tennstedt et al. 2009, Kühnlenz et al. 2016). The initial clue for this finding was provided by the different phenotypes of two AtPCS1 mutants, cad1-3 and cad1-6, under Cd and Zn excess stress (Tennstedt et al. 2009): cad1-3 is the null mutant of AtPCS1 and cad1-6 has a T-DNA insertion disrupting the C-terminal half of the Phytochelatin_C domain of AtPCS1 (Fig. 1A, B). Both mutants show the excess Zn-sensitive phenotype, but cad1-6 is relatively tolerant to Cd stress with substantial PC accumulation (Tennstedt et al. 2009, Kühnlenz et al. 2016), suggesting that the missing C-terminal region in cad1-6 is required for PCS activity triggered by Zn but not by Cd. Fig. 1 View largeDownload slide (A and B) Sequence characteristics of AtPCS1 mutants cad1-3 and cad1-6. (A) Genomic situation of AtPCS1. Black and light blue boxes indicate untranslated regions and coding regions, respectively. The sites of a point mutation (cad1-3) and T-DNA insertion (cad1-6) are indicated. (B) Domain organization of AtPCS1. The N-terminal catalytic domain (pfam05023) and the C-terminal Phytochelatin_C domain with unknown functions (DUF1984; pfam09328) are indicated. In cad1-3 there is a nucleotide substitution in exon 4 which results in an amino acid change from tryptophan to cysteine in the N-terminal catalytic domain. No AtPCS1 accumulation is observed in cad1-3 probably due to protein instability (Tennstedt et al. 2009). In cad1-6, the T-DNA insertion disrupts the coding sequence after the codon for Glu409 (Tennstedt et al. 2009), which is located in the latter half of the C-terminal domain. (C–E) Growth assay of the PC-related mutant plants under As(III) stress. (C) Phenotypes of Col-0, cad1-3, cad1-6 and abcc1/2 grown on the agar medium containing 1 or 1.5 μM As(III) for 12 d. Scale bar = 1 cm. Relative primary root length (D) and fresh weight of seedlings (E) of Col-0, cad1-3, cad1-6 and abcc1/2 plants grown on the agar medium containing different concentrations of As(III) for 12 d. Values are shown as a percentage of each control. Data represent means with the SD from two independent experiments (n = 16–24). Means sharing the same letter are not significantly different within each treatment (P < 0.05, Tukey’s HSD). Fig. 1 View largeDownload slide (A and B) Sequence characteristics of AtPCS1 mutants cad1-3 and cad1-6. (A) Genomic situation of AtPCS1. Black and light blue boxes indicate untranslated regions and coding regions, respectively. The sites of a point mutation (cad1-3) and T-DNA insertion (cad1-6) are indicated. (B) Domain organization of AtPCS1. The N-terminal catalytic domain (pfam05023) and the C-terminal Phytochelatin_C domain with unknown functions (DUF1984; pfam09328) are indicated. In cad1-3 there is a nucleotide substitution in exon 4 which results in an amino acid change from tryptophan to cysteine in the N-terminal catalytic domain. No AtPCS1 accumulation is observed in cad1-3 probably due to protein instability (Tennstedt et al. 2009). In cad1-6, the T-DNA insertion disrupts the coding sequence after the codon for Glu409 (Tennstedt et al. 2009), which is located in the latter half of the C-terminal domain. (C–E) Growth assay of the PC-related mutant plants under As(III) stress. (C) Phenotypes of Col-0, cad1-3, cad1-6 and abcc1/2 grown on the agar medium containing 1 or 1.5 μM As(III) for 12 d. Scale bar = 1 cm. Relative primary root length (D) and fresh weight of seedlings (E) of Col-0, cad1-3, cad1-6 and abcc1/2 plants grown on the agar medium containing different concentrations of As(III) for 12 d. Values are shown as a percentage of each control. Data represent means with the SD from two independent experiments (n = 16–24). Means sharing the same letter are not significantly different within each treatment (P < 0.05, Tukey’s HSD). To understand further the element-specific PCS activation mediated by the C-terminal domain, the present study examined the roles of the AtPCS1 C-terminal domain in As(III)-responsive PC synthesis first by phenotyping of cad1-6 and then by in vitro PCS activity assay. We identified two C-terminal regions specifically essential for As-dependent PCS activity and demonstrated significance of the C-terminal domain for plant As tolerance and As movement within plants. Our finding that a particular C-terminal truncation yields an AtPCS1 variant hyperactivated by As(III) may offer a chance to develop a tool useful for better restricting As accumulation in above-ground tissues. Results As(III)-sensitive phenotype of cad1-6 is comparable with that of cad1-3 We first examined the sensitivity of cad1-6 to As(III) stress in comparison with cad1-3 and the PC transporter double mutant abcc1/2 (Song et al. 2010). We employed solid agar plates containing a modified Hoagland medium established to examine toxic metal-sensitive phenotypes with reduced metal dosage compared with Murashige and Skoog (MS) medium (Tennstedt et al. 2009, Fischer et al. 2014). The growth of Col-0, cad1-3, cad1-6 and abcc1/2 was examined under a range of As(III) concentrations (Fig. 1C–E). Root and shoot growth of the three mutants were overall impaired by As(III) treatments, whereas Col-0 grew normally without exhibiting symptoms of toxicity up to a concentration of 1.5 μM As(III). The growth of known As-sensitive mutants cad1-3 and abcc1/2 were similarly inhibited by As(III) treatments (Fig. 1C, D), but the seedling weight was slightly greater in abcc1/2 than in cad1-3 (Fig. 1E), suggesting that our sensitivity assay can detect slight phenotypic differences of As(III) sensitivity. Importantly, cad1-6 showed phenotypes very similar to those of cad1-3 under both moderate (1 μM) and higher (1.5 μM) As(III) exposure (Fig. 1C). Purple-colored leaves were observed in both cad1-3 and cad1-6 exposed to 1.5 μM As(III), but not in Col-0 and abcc1/2 (Fig. 1C). The comparable growth retardation of cad1-3 and cad1-6 exposed to As(III) was evident when root length (Fig. 1D) and seedling fresh weight were measured (Fig. 1E): growth of the two AtPCS1 mutants was similarly inhibited by both moderate (0.75 and 1 μM) and relatively high (1.5 μM) As(III) treatments. Taken together, the results of the growth assays under conditions allowing sensitive detection of toxicity effects in the presence of low metal/metalloid doses demonstrated that there is little difference in As(III) sensitivity between cad1-3 and cad1-6. Distribution of As and essential minerals within plants is altered similarly in cad1-3 and cad1-6 For the second phenotype of the mutants, elemental distribution in plants exposed to As(III) was examined. As and essential element concentrations in the roots and shoots were analyzed and then elemental partitioning between shoots and roots was calculated. The As concentrations in shoots and roots of cad1-3 and cad1-6 were similarly different from those of Col-0 (Fig. 2A, B). Distribution of As to shoots (shoot/root ratio) was significantly and equally higher in cad1-3 and cad1-6 than in Col-0 (Fig. 2C). abcc1/2 also showed increased As distribution to shoots compared with Col-0 (Fig. 2C). Fig. 2 View largeDownload slide Arsenic concentrations in shoots (A) and roots (B), and shoot/root ratios of As (C) in the PC pathway mutants under As(III) stress. Col-0, cad1-3, cad1-6 and abcc1/2 were grown on the control agar medium for 10 d and then transferred to the medium containing 5 μM As(III). After 4 d, plants were harvested and As concentrations in roots and shoots were separately quantified by ICP-OES. Data represent means with the SD from three independent experiments (n = 10). Means sharing the same letter are not significantly different (P < 0.05, Tukey’s HSD). Fig. 2 View largeDownload slide Arsenic concentrations in shoots (A) and roots (B), and shoot/root ratios of As (C) in the PC pathway mutants under As(III) stress. Col-0, cad1-3, cad1-6 and abcc1/2 were grown on the control agar medium for 10 d and then transferred to the medium containing 5 μM As(III). After 4 d, plants were harvested and As concentrations in roots and shoots were separately quantified by ICP-OES. Data represent means with the SD from three independent experiments (n = 10). Means sharing the same letter are not significantly different (P < 0.05, Tukey’s HSD). Among essential metals (Fe, Mn, Cu and Zn), significant alterations of Zn accumulation and distribution were observed in the three tested mutants when treated with As(III) (Fig. 3). Under As(III) treatment, Zn distribution to the shoots of the three mutants dropped down to 30% of that of Col-0, derived from both an increase of Zn accumulation in the roots and a decrease in the shoots of the mutants. Such a difference in the Zn shoot/root ratio between the mutants and Col-0 was not observed under control conditions, although both cad1-3 and cad1-6 showed slightly yet significantly reduced shoot Zn concentrations compared with Col-0. In contrast to Zn, there was no significant difference for tissue concentrations and shoot/root ratios of Fe, Mn and Cu among cad1-3, cad1-6 and Col-0 under both control and As(III) conditions. abcc1/2 showed significantly lower concentrations of Fe and Mn in the roots and shoots under As(III) treatment, compared with Col-0 (Fig. 3). Fig. 3 View largeDownload slide Essential microelement concentrations in shoots and roots, and shoot/root ratios in the PC pathway mutants under normal conditions and As(III)-stressed conditions. Col-0, cad1-3, cad1-6 and abcc1/2 were grown on the control agar medium for 10 d and then transferred to the control medium or medium containing 5 μM As(III). After 4 d, plants were harvested, and element concentrations in roots and shoots were separately quantified by ICP-OES. Data represent means with the SD from two independent experiments (n = 5–6) for control conditions and three independent experiments (n = 7–10). Means sharing the same letter are not significantly different within each treatment (P < 0.05, Tukey’s HSD). Fig. 3 View largeDownload slide Essential microelement concentrations in shoots and roots, and shoot/root ratios in the PC pathway mutants under normal conditions and As(III)-stressed conditions. Col-0, cad1-3, cad1-6 and abcc1/2 were grown on the control agar medium for 10 d and then transferred to the control medium or medium containing 5 μM As(III). After 4 d, plants were harvested, and element concentrations in roots and shoots were separately quantified by ICP-OES. Data represent means with the SD from two independent experiments (n = 5–6) for control conditions and three independent experiments (n = 7–10). Means sharing the same letter are not significantly different within each treatment (P < 0.05, Tukey’s HSD). Cysteine and GSH accumulation is altered similarly in cad1-3 and cad1-6 Cysteine and GSH concentrations in the PC-related mutant plants exposed to As(III) were measured (Fig. 4). abcc1/2 basically showed Col-0-like patterns of cysteine and GSH concentrations in both roots and shoots. On the other hand, cysteine and GSH concentrations of cad1-6 and cad1-3 were significantly, and to a similar degree, elevated in both roots (Fig. 4A, B) and shoots (Fig. 4C, D) compared with Col-0. The difference between the AtPCS1 mutants and Col-0 was evident in roots, with 3-fold higher concentrations in the mutants (Fig. 4A, B). Fig. 4 View largeDownload slide Thiol concentrations in the PC pathway mutants under As(III) stress. Col-0, cad1-3, cad1-6 and abcc1/2 were grown on the control agar medium for 10 d and then transferred to the medium containing 5 μM As(III). After 4 d, plants were harvested and cysteine (A and C) and GSH (B and D) concentrations in roots (A and B) and shoots (C and D) were quantified by HPLC after thiol extraction and derivatization. Data represent means with the SD from at least two independent experiments (n = 6). Means sharing the same letter are not significantly different (P < 0.05, Tukey’s HSD). Fig. 4 View largeDownload slide Thiol concentrations in the PC pathway mutants under As(III) stress. Col-0, cad1-3, cad1-6 and abcc1/2 were grown on the control agar medium for 10 d and then transferred to the medium containing 5 μM As(III). After 4 d, plants were harvested and cysteine (A and C) and GSH (B and D) concentrations in roots (A and B) and shoots (C and D) were quantified by HPLC after thiol extraction and derivatization. Data represent means with the SD from at least two independent experiments (n = 6). Means sharing the same letter are not significantly different (P < 0.05, Tukey’s HSD). Defect of PCS activity in cad1-6 exposed to As(III) The results of phenotyping cad1-6 under As(III) exposure suggested that cad1-6 responds to As(III) stress very similarly to cad1-3. Such cad1-3-like phenotypes of cad1-6 further indicated a possibility that PCS activity of cad1-6 in response to As(III) is mostly impaired. We thus measured PC2 and PC3 concentrations in the plants exposed to As(III) (Fig. 5). In roots, Col-0 accumulated the highest amounts of PC2 and PC3, followed by abcc1/2 (Fig. 5A, B). Under this condition, cad1-3 roots showed trace PC2 and PC3 accumulation. The PC3 concentration in cad1-6 roots was also under the limit of quantification of our HPLC system (Fig. 5B), and only very slight PC2 accumulation was detected in cad1-6 roots (Fig. 5A). In shoots, no detectable PCs were found in either cad1-3 or cad1-6, whereas Col-0 and abcc1/2 accumulated substantial amounts of PC2 and PC3 (Fig. 5C, D). Fig. 5 View largeDownload slide PC concentrations in the PC pathway mutants under As(III) stress. Col-0, cad1-3, cad1-6 and abcc1/2 were grown on the control agar medium for 10 d and then transferred to the medium containing 5 μM As(III). After 4 d, plants were harvested and PC2 (A and C) and PC3 (B and D) concentrations in roots (A and B) and shoots (C and D) were quantified by HPLC after thiol extraction and derivatization. Data represent means with the SD from at least two independent experiments (n = 4–6). Means sharing the same letter are not significantly different (P < 0.05, Tukey’s HSD). Fig. 5 View largeDownload slide PC concentrations in the PC pathway mutants under As(III) stress. Col-0, cad1-3, cad1-6 and abcc1/2 were grown on the control agar medium for 10 d and then transferred to the medium containing 5 μM As(III). After 4 d, plants were harvested and PC2 (A and C) and PC3 (B and D) concentrations in roots (A and B) and shoots (C and D) were quantified by HPLC after thiol extraction and derivatization. Data represent means with the SD from at least two independent experiments (n = 4–6). Means sharing the same letter are not significantly different (P < 0.05, Tukey’s HSD). As(III)-dependent PCS activity of C-terminally truncated AtPCS1 mutants in the fission yeast expression system To examine further the roles of the C-terminal regions of AtPCS1, we analyzed As(III)-dependent PCS activity of the AtPCS1 deletion series using the fission yeast expression system (Kühnlenz et al. 2016). In addition to the wild-type full-length AtPCS1, four different truncated AtPCS1 mutants were examined (Fig. 6A). In a previous study, expression of these mutants in the Schizosaccharomyces pombe PCS knockout strain Δpcs (Clemens et al. 1999) demonstrated differential responses of the mutant proteins to Cd and Zn exposure, and suggested respective protein parts as being required for the PCS activity responding to Cd or Zn (Kühnlenz et al. 2016). We first confirmed the expression of the wild-type AtPCS1 and four truncated variants in the respective cell lines exposed to As(III) (Supplementary Fig. S1). Using this established system, the PCS activity was measured by exposing the cells to As(III). PC2 and PC3 were substantially synthesized in the cells with the wild-type AtPCS1, whereas both PC2 and PC3 levels in the cells with the empty vector were under the limit of quantification of our analytical system (Fig. 6B). Under this condition, cells expressing the shortest truncated mutant Δ373–485 did not accumulate PC2 and PC3 above the quantification levels like cells carrying the empty vector (Fig. 6B). Cells expressing longer mutants Δ460–485 and Δ471–485 showed statistically similar PC2 and PC3 accumulation: the mutants accumulated approximately 25–50% of PC2 and 60% of PC3 compared with the wild-type AtPCS1-expressing cells. Interestingly, expression of the longest version, Δ476–485, resulted in cells accumulating higher levels of PC2 and PC3 compared with the cells with wild-type AtPCS1. Fig. 6 View largeDownload slide PCS activity assay of AtPCS1 deletion mutants of the C-terminal domain in response to As(III). (A) Schematic of full-length AtPCS1 and a series of C-terminally truncated AtPCS1s expressed in the S. pombe PCS knockout strain Δpcs. (B) PC concentrations in the S. pombe cells exposed to 10 μM As(III) for 4 h. PC2 and PC3 were quantified by HPLC after thiol extraction from the harvested cells and derivatization. Data represent means with the SD from two independent experiments (n = 4). Means sharing the same letter are not significantly different (P < 0.05, Tukey’s HSD). (C) Growth of the S. pombe Δpcs cells harboring an empty vector pSGP72 or expressing AtPCS1 or truncated variants exposed to As(III). Data represent means with the SD from two independent experiments (n = 8). Means sharing the same letter are not significantly different within each treatment (P < 0.05, Tukey’s HSD). Fig. 6 View largeDownload slide PCS activity assay of AtPCS1 deletion mutants of the C-terminal domain in response to As(III). (A) Schematic of full-length AtPCS1 and a series of C-terminally truncated AtPCS1s expressed in the S. pombe PCS knockout strain Δpcs. (B) PC concentrations in the S. pombe cells exposed to 10 μM As(III) for 4 h. PC2 and PC3 were quantified by HPLC after thiol extraction from the harvested cells and derivatization. Data represent means with the SD from two independent experiments (n = 4). Means sharing the same letter are not significantly different (P < 0.05, Tukey’s HSD). (C) Growth of the S. pombe Δpcs cells harboring an empty vector pSGP72 or expressing AtPCS1 or truncated variants exposed to As(III). Data represent means with the SD from two independent experiments (n = 8). Means sharing the same letter are not significantly different within each treatment (P < 0.05, Tukey’s HSD). We then examined growth of the cells expressing AtPCS1 or the truncated variants under different concentrations of As(III) (Fig. 6C). The cells expressing the full-length AtPCS1 showed about 2-fold better growth than control cells with an empty vector under both 25 and 50 μM As(III). Under these conditions, growth of the cells expressing the shortest truncated mutant Δ373–485 was statistically equal to that of empty vector controls, whereas the cells expressing Δ460–485 or Δ471–485 showed intermediate tolerance to As(III). The cells expressing Δ476–485 represented slightly yet significantly better growth under both As(III) conditions compared with the cells expressing the wild-type AtPCS1 (Fig. 6C). Enhanced PCS activity of Δ476–485 recombinant protein in response to As(III) To examine further the As(III)-dependent PCS activity of Δ476–485 by the in vitro assay (Fischer et al. 2014), we purified the recombinant proteins of Δ476–485 and the wild-type AtPCS1 (Supplementary Fig. S2A). As(III)-dependent PCS activities of the recombinant proteins were assessed relative to Cd-dependent activities of the respective proteins as previously described (Oven et al. 2002, Fischer et al. 2014). Relative As(III)-dependent PCS activity of the wild-type protein in the presence of 100 μM As(III) was 19% of the activity in the presence of 100 μM Cd (Supplementary Fig. S2B), which was very similar to the reported data for purified AtPCS1 (Oven et al. 2002). Compared with the wild-type AtPCS1, Δ476–485 clearly showed much higher As(III)-dependent PCS activity relative to Cd-dependent activity. This difference was also observed when 10 μM As(III) or Cd were added to the assay mixtures. Discussion Due to human activities or geochemical factors, phytoavailability of several elements such as Cd, As, Zn and aluminum (Al) in soils can reach levels toxic to plants. Since plants are sessile, they have to cope with such elemental toxicity in the rhizosphere. Molecular mechanisms underlying plant tolerance against soil-derived elemental stress have been revealed for several cases, and many of the identified proteins are generally associated with a single elemental stress. For example, AtMTP1, a tonoplast Zn transporter, confers the excess Zn tolerance to A. thaliana (Kobae et al. 2004, Desbrosses-Fonrouge et al. 2005); however, AtMTP1 is probably not involved in Cd, nickel (Ni) and manganese (Mn) tolerance (Kobae et al. 2004, Weber et al. 2013). In contrast, the PC/PCS system is associated with detoxification of a wide range of elemental toxicity. Analyses of cad1-3 and abcc1/2 suggested the significant role of PC synthesis and subsequent vacuolar compartmentation in detoxifying toxic inorganic ions such as Cd, Hg and As (Howden et al. 1995, Ha et al. 1999, Song et al. 2010, Park et al. 2011). AtPCS1 also plays an important role in detoxification of Pb and excess Zn (Tennstedt et al. 2009, Fischer et al. 2014). Various other elements including Cu, silver (Ag) and antimony (Sb) are also able to induce PC synthesis in vitro, but iron (Fe) and Mn are not (Grill et al. 1987, Cazale and Clemens 2001, Kühnlenz et al. 2014). Such responsiveness of PCS to a relatively wide range of elements raised the question as to how PCS is activated by these different elements. It was suggested that the AtPCS1 C-terminal region is involved in AtPCS1 activation by different metals such as Cd and Zn (Ruotolo et al. 2004, Tennstedt et al. 2009, Kühnlenz et al. 2016). However, mechanisms of As-dependent AtPCS1 activation have been little examined, whereas the physiological functions of PCS/PC have been well demonstrated in regard to As exposure. Moreover, understanding mechanisms of As-dependent PCS activation could be potentially applied for molecular breeding of PCS variants hyperactivated by As(III). For Cd, a previous study conducted site-directed mutagenesis of AtPCS1 and identified variants which enhance Cd tolerance (Cahoon et al. 2015). The present study aimed to understand roles of the AtPCS1 C-terminal domain in As(III) responses and search for variants with enhanced As(III)-dependent PCS activity. We first characterized phenotypes of cad1-6 in comparison with the null mutant cad1-3 under As(III) stress conditions. cad1-6 has a T-DNA insertion in exon 8 of AtPCS1, disrupting the coding sequence after the codon for Glu409 (Fig. 1A, B) (Tennstedt et al. 2009). Due to the T-DNA insertion, cad1-6 is suggested to express a C-terminally truncated AtPCS1 (Tennstedt et al. 2009, Kühnlenz et al. 2016). To examine the As(III) sensitivity of cad1-6, we employed the growth assay which was able to detect slight differences of growth between cad1-3 and abcc1/2. Under all As(III) treatments tested, the phenotypes of cad1-6 closely resembled those of cad1-3: there were no significant differences in root length (Fig. 1D) and plant fresh weight between the AtPCS1 mutants (Fig. 1E). Both AtPCS1 mutants accumulated anthocyanin in shoots under 1.5 μM As(III) (Fig. 1C), whereas abcc1/2 did not show such pigment accumulation. The cad1-3 like As sensitivity of cad1-6 suggested that PCS activity in response to As(III) is diminished in cad1-6 to the level of cad1-3. Similarly, As accumulation (Fig. 2) as well as cysteine and GSH concentrations (Fig. 4) in cad1-6 were also equal to those of cad1-3 under As(III) treatment, further supporting the hypothesis. Concerning As accumulation, the distribution of As to the shoot was significantly higher in cad1-6 and cad1-3 compared with the wild type Col-0 when exposed to As(III) (Fig. 2C). A previous study also reported increased As translocation to shoots of cad1-3 exposed to As(V) and suggested that AtPCS1-mediated PC synthesis in response to As facilitates vacuolar compartmentation of PC–As complexes in root cells and thus retains As in the roots (Liu et al. 2010). Increased As distribution in cad1-6 shoots comparable with cad1-3 may explain the stronger toxicity symptoms of cad1-6 and cad1-3 shoots relative to abcc1/2 (Fig. 1C), and suggests a defect of PC synthesis and PC–As complex formation in cad1-6 exposed to As(III). In line with the hypothesis, in cad1-6 exposed to As(III), only trace PCs were detected, as found for cad1-3, both in shoots and in roots (Fig. 5), demonstrating that As(III)-dependent PCS activity is impaired in cad1-6 to the same extent as in the null mutant cad1-3. Likewise, abolished AtPCS1 activity of cad1-6 in response to As(III) is reflected in increased GSH and cysteine accumulation (Fig. 4). Similar trace PC accumulation was observed when the AtPCS1 mutant plants were exposed to excess Zn, whereas Cd exposure led to substantial PC2 accumulation in cad1-6 roots (Tennstedt et al. 2009). The different PC accumulation between cad1-6 and cad1-3 exposed to Zn or Cd suggested that C-terminally truncated AtPCS1 expression expected in cad1-6 can be activated by Cd but not by Zn (Tennstedt et al. 2009). Indeed, in vitro experiments and heterologous expression suggested that the stretch between His460 and Arg470 of AtPCS1 is as an essential C-terminal region for activation by Zn, but for Cd-triggered activation the regions between Glu283 and Asp372 as well as the N-terminal domain are crucial (Ruotolo et al. 2004, Romanyuk et al. 2006, Kühnlenz et al. 2016). The cad1-3-like phenotypes of cad1-6 under As(III) stress demonstrated in this study (Figs. 1–5) indicate a significant role for the AtPCS1 C-terminal domain in As-dependent PCS activity as is the case for Zn excess-dependent activation. To examine this hypothesis further, a series of AtPCS1 C-terminal deletion mutants (Kühnlenz et al. 2016; Fig. 6A) was expressed in the fission yeast expression system (Supplementary Fig. S1), and their PCS activities in response to As(III) were examined. The C-terminal region spanning Asp373 to Leu459 was suggested to be crucial for the activation by As(III) (Fig. 7), since the shortest mutant Δ373–485 did not show any PC synthesis activity above the background level of the empty vector control, while a longer mutant Δ460–485 was able to synthesize an appreciable amount of PCs (Fig. 6B). The stretch between His460 and Arg470 essential for activation by Zn (Kühnlenz et al. 2016) is less likely to be important for activation by As(III), because there was no significant effect of adding the stretch from His460 to Arg470 when comparing the activities of Δ460–485 and Δ471–485. Cys471 to Lys475 was suggested as the second crucial amino acid stretch for activation by As(III) (Fig. 7) based on the remarkable increase of PC accumulation in Δ476–485 compared with the shorter mutants. Considering that the As(III)-dependent PC accumulation in Δ476–485 exceeded even that of the wild-type AtPCS1 (Fig. 6B; Supplementary Fig. S2) which conferred additional As(III) tolerance to the cells (Fig. 6C), it is also possible that the amino acid residues after Glu476 may negatively affect the PCS activation by As(III). Fig. 7 View largeDownload slide Partial amino acid sequences of the AtPCS1 C-terminal domain. The two regions suggested to be important for As(III)-dependent activation in the present study are indicated (Asp373 to Leu459 and Cys471 to Lys475). The regions for Cd- or Zn-dependent activation are also indicated based on the results of Kühnlenz et al. (2016). Fig. 7 View largeDownload slide Partial amino acid sequences of the AtPCS1 C-terminal domain. The two regions suggested to be important for As(III)-dependent activation in the present study are indicated (Asp373 to Leu459 and Cys471 to Lys475). The regions for Cd- or Zn-dependent activation are also indicated based on the results of Kühnlenz et al. (2016). Overall, the deletion analysis of the AtPCS1 C-terminal domain suggests at least two regions important for AtPCS1 activation by As(III): Asp373 to Leu459 and Cys471 to Lys475 (Fig. 7). The former region contains Glu409, after which the coding sequence is disrupted by the T-DNA insertion in cad1-6 (Fig. 1B) (Tennstedt et al. 2009). Taken together with the abolished PCS activity in cad1-6 exposed to As(III) (Fig. 5), it may be possible to narrow down the candidate region to the sequence from after Glu409 to Leu459. Compared with the suggested regions in the C-terminal domain important for AtPCS1 activation by Cd and excess Zn (Ruotolo et al. 2004, Kühnlenz et al. 2016), we would suggest that the two regions for As-dependent activation are unique (Fig. 7). Through the phenotyping of the PC-related mutant plants exposed to As(III), we observed the drastic reduction of Zn accumulation in the AtPCS1 mutants exposed to As(III) (Fig. 3). Such a reduction of the Zn shoot/root ratio was not observed under control conditions; however, cad1-3 and cad1-6 showed slight yet significant reduction of Zn concentrations in shoots. A previous study suggested that AtPCS1 controls Zn delivery from roots to shoots under normal Zn conditions (Kühnlenz et al. 2016). Our results support the idea and further suggest that the contribution of AtPCS1-dependent Zn delivery to shoots is more evident under As(III) stress. Enhanced PC production triggered by As(III) in root cells may facilitate PC–Zn complex formation for translocation to shoots and to maintain Zn levels in shoots. Alternatively, other PC-independent Zn translocation pathways are affected in As(III)-exposed plants, which would increase the importance of PC-dependent Zn mobility. The same Zn reduction was observed for abcc1/2 mutant plants, confirming that alteration of PC metabolism and/or intracellular transport may also affect Zn homeostasis. It should be noted that abcc1/2 also showed reduced accumulation of Fe and Mn in the roots and shoots (Fig. 3). In conclusion, we suggest the that the two unique stretches of the AtPCS1 C-terminal region are crucial for As(III)-dependent activation. The analyses of cad1-6 suggest that the C-terminal regions are crucial for in planta PC synthesis in response to As(III) stress and eventually for As(III) detoxification and As mobility within plants. It should be further investigated which amino acid residues are the keys for As(III)-dependent AtPCS1 activation in vitro and in planta as well. Cysteine is established as the major amino acid binding to As(III) (Zhou et al. 2011, Shen et al. 2013). Hydrophilic amino acids such as arginine and aspartate are also suggested as important residues to form As-binding sites together with cysteine (Bennett et al. 2001, Martin et al. 2001). The two identified regions of AtPCS1 crucial for As(III)-dependent activation include cysteine, and the longer stretch Asp373 to Leu459 contains arginine and aspartate residues. Such amino acid residues within the regions would be primary candidates mediating As(III)-dependent AtPCS1 activation. It should also be noted that the C-terminal regions are highly conserved among higher plant PCSs (Kühnlenz et al. 2016). Another interesting finding is that the Δ476–485 variant which lacks the C-terminal 10 amino acid residues shows enhanced PCS activity compared with the wild-type protein in response to As(III). Since PCS contributes to restricting As mobility in plants (Liu et al. 2010, Hayashi et al. 2017, Uraguchi et al. 2017), this variant could potentially be used as a tool to decrease As distribution to seeds or grains. Materials and Methods Plant materials and growth conditions Arabidopsis thaliana wild type (Col-0), the AtPCS1 null mutant cad1-3 (Howden et al. 1995), the AtPCS1 T-DNA insertion line cad1-6 (Tennstedt et al. 2009) and the AtABCC1 and AtABCC2 double knockout mutant line abcc1/2 (Song et al. 2010) were used in this study. For As(III) sensitivity assay, agar plates containing 1/10th modified Hoagland medium were used for plant cultivation (Tennstedt et al. 2009, Fischer et al. 2014) [100 μM (NH4)2HPO4, 200 μM MgSO4, 280 μM Ca(NO3)2, 600 μM KNO3, 5 μM Fe-HBED, 1% (w/v) sucrose, 5 mM MES, 1% (w/v) purified agar (Nacalai Tesque), pH 5.7]. When cultivating plants for elemental and PC analyses, the following microelements were additionally supplemented to the medium (4.63 μM H3BO3, 32 nM CuSO4, 915 nM MnCl2, 77 nM ZnSO4, 11 nM MoO3) (Kühnlenz et al. 2014). Arabidopsis seeds were surface sterilized and sown on agar plates. After 2 d stratification at 4°C, plants were grown vertically in a growth chamber (16 h light/8 h dark, 22°C) as described elsewhere. As(III) sensitivity assay To examine the sensitivity of Arabidopsis to As(III), plants were grown for 12 d on agar plates containing different concentrations of As(III) as NaAsO2 (0.75, 1 and 1.5 μM). The plates without addition of As(III) served as controls. Plant growth was assessed by primary root length and seedling fresh weight measurements at the end of the cultivation. Elemental analysis For elemental analyses of plant samples, plants were grown on the control plates (solidified with 1.5% agar) for 10 d. Uniformly grown seedlings were then transferred to control plates or plates containing 5 μM As (III) and incubated for an additional 4 d. Roots and shoots from each plate (normally 15 seedlings per genotype) were separately pooled as a single sample. Shoot samples were washed with MilliQ water twice. Root samples were subjected to sequential washing procedures: roots were desorbed for 10 min each in ice-cold MilliQ water, 20 mM CaCl2 (twice), 10 mM EDTA (pH 5.7) and MilliQ water. Harvested roots and shoots were dried at 50°C before acid digestion. Dried plant samples were wet-digested with HNO3. Elemental concentrations in the samples were quantified by inductively coupled plasma-optical emission spectroscopy (ICP-OES; iCAP7400Duo, Thermo-Fisher Scientific). PC analysis of plant samples For PC analyses of plant samples, plants were grown on the control plates (solidified with 1% agar) for 10 d. Uniformly grown seedlings were then transferred to plates containing 5 μM As(III) and incubated for an additional 4 d. Roots and shoots from each plate (normally 15 seedlings per genotype) were separately pooled as a single sample. The plant samples were frozen in liquid nitrogen after fresh weight measurement. Homogenously ground material was used for thiol extraction. Thiols were extracted and derivatized as described, with a slight modification (Kühnlenz et al. 2014). A 10 mg aliquot of plant material was extracted with 30 μl of 0.1% (v/v) trifluoroacetic acid (TFA) containing 6.3 mM diethylenetriaminepentaacetic acid (DTPA). Thiol derivatives were analyzed by HPLC equipped with a fluorescence detector (Nishida et al. 2016). Quantification of thiol derivatives was performed via standards of cysteine, GSH, PC2 and PC3 after normalization to the internal N-acetylcysteine (NAC) standard. Standards of PC2 and PC3 were synthesized by Bonac Corporation (http://www.bonac.com). Heterologous expression of AtPCS1 and the truncated variants in fission yeast For the in vivo testing of PCS variants, the fission yeast (S. pombe) PCS knockout strain Δpcs (Clemens et al. 1999) heterologously expressing HA-tagged AtPCS1 or a series of C-terminal deletion mutants were used (Kühnlenz et al. 2016). Cells carrying the empty vector pSGP72 served as negative control. The assay was conducted as described previously, with some modifications (Kühnlenz et al. 2016). Yeast cultivation was carried out at 30°C in Edinburgh’s minimal medium (EMM) supplemented with 1 μM thiamine. Pre-cultured cells were inoculated to an OD600 = 0.1 in EMM supplemented with 1 μM thiamine and grown overnight. Then cells were inoculated at an OD600 = 0.5 in EMM supplemented with 1 μM thiamine and 10 μM As(III). After 4 h incubation, the OD600 was measured for each culture to assess cell numbers. Cells were then harvested and frozen in liquid N2 prior to protein and PC extraction. For detection of the HA-tagged AtPCS1 and truncated proteins, protein extracts were prepared from the harvested cells as described, with minor modifications (Matsuo et al. 2006), and were subjected to Western blotting using the anti-HA tag antibody (MBL, M180-3) as the primary antibody. Horseradish peroxidase-conjugated goat anti-mouse IgG (GE Healthcare, NA931V) was used as the secondary antibody and Chemi Lumi One L (Nacalai Tesque) was used for detection. For PC quantification, the extraction buffer [0.1% (v/v) TFA containing 6.3 mM DTPA] was used for thiol extraction from the harvested cells. Thiol derivatization and quantification by HPLC were performed as described elsewhere. For growth assay, pre-cultured cells were prepared using EMM supplemented with 1 μM thiamine. Then cells were inoculated at an OD600 = 0.1 in EMM supplemented with 1 μM thiamine and two different concentrations of As(III) (25 and 50 μM). Growth of the cells was monitored by measuring the OD600 for 24 h. Recombinant protein production and PCS activity assay Sequences coding for the full-length AtPCS1 protein and the truncated variant Δ476–485 were amplified from cDNA using the primers listed in Supplementary Table S1 and cloned into the expression vector pET19TEV in-frame with an N-terminal 6×His tag. The expression plasmids were transformed into Escherichia coli Rosetta2(DE3)pLysS cells. Expression was induced at an OD600 of about 0.6 by addition of 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Cells were grown for 14 h at 21°C and 150 r.p.m., after which they were harvested (25 min, 4°C, 6,160×g). Soluble His-tagged recombinant protein was purified using an Ni-NTA matrix (Qiagen) as previously described (Fischer et al. 2014). Protein concentrations were determined using Roti®-Quant (Carl Roth) according to Bradford, and estimated based on a bovine serum albumin (BSA) dilution series following SDS–PAGE. PCS activity was assessed by adding heterologously expressed protein in 20 μl of storage buffer [50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 7.0, 25% (v/v) glycerol] to 80 μl of activity buffer [50 mM HEPES pH 7.0, 12.5 mM GSH, 10% (v/v) glycerol]. Enzymes were activated by addition of Cd(II) or As(III) at the indicated concentrations. The reaction was allowed to proceed for up to 60 min at 35°C and stopped by addition of 1.1 μl of 10% (v/v) TFA. Thiols were extracted, monobromobimane-derivatized and PC2 was quantified by HPLC as described (Kühnlenz et al. 2014). Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the Japan Society for the Promotion of Science [grant No. 16K14873 to S.U.] and the Deutsche Forschungsgemeinschaft [CL 152/7-2 to S.C.]. Acknowledgments We thank Youngsook Lee (POSTECH) and Enrico Martinoia (University of Zurich) for providing abcc1/2 seeds, and Silke Matros for excellent technical assistance. Standards of PCs were kindly provided by Shin-ichi Nakamura (Tokyo University of Agriculture). Disclosures The authors have no conflicts of interest to declare. References Bennett M.S., Guan Z., Laurberg M., Su X.D. ( 2001) Bacillus subtilis arsenate reductase is structurally and functionally similar to low molecular weight protein tyrosine phosphatases. Proc. Natl. Acad. Sci. USA  98: 13577– 13582. 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Identification of C-terminal Regions in Arabidopsis thaliana Phytochelatin Synthase 1 Specifically Involved in Activation by Arsenite

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© The Author(s) 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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Abstract

Abstract Phytochelatins (PCs) are major chelators of toxic elements including inorganic arsenic (As) in plant cells. Their synthesis confers tolerance and influences within-plant mobility. Previous studies had shown that various metal/metalloid ions differentially activate PC synthesis. Here we identified C-terminal parts involved in arsenite- [As(III)] dependent activation of AtPCS1, the primary Arabidopsis PC synthase. The T-DNA insertion in the AtPCS1 mutant cad1-6 causes a truncation in the C-terminal regulatory domain that differentially affects activation by cadmium (Cd) and zinc (Zn). Comparisons of cad1-6 with the AtPCS1 null mutant cad1-3 and the double mutant of tonoplast PC transporters abcc1/2 revealed As(III) hypersensitivity of cad1-6 equal to that of cad1-3. Both cad1-6 and cad1-3 showed increased As distribution to shoots compared with Col-0, whereas Zn accumulation in shoots was equally lower in cad1-6 and cad1-3. Supporting these phenotypes of cad1-6, PC accumulation in the As(III)-exposed plants were at trace level in both cad1-6 and cad1-3, suggesting that the truncated AtPCS1 of cad1-6 is defective in PCS activity in response to As(III). Analysis of a C-terminal deletion series of AtPCS1 using the PCS-deficient mutant of fission yeast suggested important regions within the C-terminal domain for As(III)-dependent PC synthesis, which were different from the regions previously suggested for Cd- or Zn-dependent activation. Interestingly, we identified a truncated variant more strongly activated than the wild-type protein. This variant could potentially be used as a tool to better restrict As mobility in plants. Introduction Arsenic (As) is ranked first on the US Agency for Toxic Substances and Disease Registry (ATSDR) 2013 Priority List of Hazardous Substances (http://www.atsdr.cdc.gov/spl). Long-term exposure to low levels of As has been associated with various human diseases including cancer and cardiovascular disease, as well as higher overall mortality (Jomova et al. 2011). Thus As accumulation in plants harbors a potential risk to human health as a source of As intake (Meharg 2004, Gilbert-Diamond et al. 2011). Control over As accumulation in plants is needed to achieve mitigation of human As exposure through plant-derived food ingestion. Inorganic forms of As, namely arsenite [As(III)] and arsenate [As(V)], are more toxic to organisms than organic forms such as arsenosugar and arsenobetaine. Plants absorb both As(III) and As(V) through root-expressed members of the aquaporin (Ma et al. 2008, Kamiya et al. 2009, Xu et al. 2015) and phosphate transporter families , respectively (Shin et al. 2004, Wu et al. 2011, Kamiya et al. 2013). Absorbed As(V) is rapidly reduced to As(III) by arsenate reductases in root cells (Chao et al. 2014, Sánchez-Bermejo et al. 2014, Shi et al. 2016, Xu et al. 2017). Therefore plant cells are challenged mainly by As(III) after exposure to either As(V) or As(III). As(III) toxicity has been attributed to its high affinity for sulfhydryl groups. As(III) can bind to reduced cysteines in peptides and proteins such as zinc-finger proteins (Zhao et al. 2012, Shen et al. 2013). Glutathione (GSH), a major cysteine-containing peptide in cells, can form a complex with As(III) in a molar ratio of 3 : 1 (Scott et al. 1993, Spuches et al. 2005). In plant cells, phytochelatins (PCs) are synthesized by PC synthases (PCSs) from GSH when plants are exposed to non-essential toxic ions as well as essential metals such as zinc (Zn) and copper (Cu). PCs are polypeptides with the general structure (γ-Glu–Cys)n–Gly (usually n = 2–7, referred to as PC2–PC7) and are major chelators of toxic elements including inorganic As in plant cells. For As(III), it has been demonstrated in vitro that a PC2–As(III) complex is formed with three of four cysteines in two molecules of PC2 (Schmöger et al. 2000). Various PC–As species have been detected in plants (Liu et al. 2010) and the chelation of As(III) with PCs is crucial for As(III) detoxification in the plant cells. In Arabidopsis, AtPCS1 is the primary PCS synthesizing PCs in response to toxic inorganic ions of As, cadmium (Cd), mercury (Hg) and lead (Pb) (Howden et al. 1995, Ha et al. 1999, Fischer et al. 2014) as well as under conditions of Zn excess (Tennstedt et al. 2009). The AtPCS1 null mutant cad1-3 displays enhanced sensitivity to these toxic elements including As, suggesting a central role for AtPCS1 in toxic element detoxification. The PC–metal(loid) complexes are sequestered into the vacuoles by tonoplast PC transporters AtABCC1 and AtABCC2 (Song et al. 2010, Park et al. 2011). The rice ortholog OsABCC1 is correspondingly responsible for As–PC complex sequestration in vacuoles (Song et al. 2014). Thus, both PC synthesis and transport are crucial steps for achieving PC-dependent detoxification of toxic elements in plant cells. Another important role indicated for PC synthesis is its involvement in long-distance element transport within plants. For example, in rice, PC synthesis by OsPCS1 and 2 and vacuolar compartmentation of PC–As complexes mediated by OsABCC1 significantly retard long-distance As transport from root to shoot and further to grains (Song et al. 2014, Hayashi et al. 2017, Uraguchi et al. 2017). There are also some indications in Arabidopsis that AtPCS1-dependent PC synthesis controls long-distance transport of As as well as Cd and Zn (Chen et al. 2006, Liu et al. 2010, Kühnlenz et al. 2016). Despite these physiological functions of AtPCS1, especially in As detoxification and transport, mechanisms governing AtPCS1 activation by As remain to be elucidated. Higher plant PCSs have a general structure with two major domains (Rea 2012): the N-terminal catalytic domain (pfam05023) and the C-terminal Phytochelatin_C domain with unknown functions (pfam09328). In vitro experiments indicated that in addition to the N-terminal domain, different regions of the AtPCS1 C-terminal domain are crucial for metal-specific PCS activation (Ruotolo et al. 2004, Romanyuk et al. 2006). Recently it was also suggested that in planta Cd- and Zn-dependent PC synthesis require different regions of the AtPCS1 C-terminal domain (Tennstedt et al. 2009, Kühnlenz et al. 2016). The initial clue for this finding was provided by the different phenotypes of two AtPCS1 mutants, cad1-3 and cad1-6, under Cd and Zn excess stress (Tennstedt et al. 2009): cad1-3 is the null mutant of AtPCS1 and cad1-6 has a T-DNA insertion disrupting the C-terminal half of the Phytochelatin_C domain of AtPCS1 (Fig. 1A, B). Both mutants show the excess Zn-sensitive phenotype, but cad1-6 is relatively tolerant to Cd stress with substantial PC accumulation (Tennstedt et al. 2009, Kühnlenz et al. 2016), suggesting that the missing C-terminal region in cad1-6 is required for PCS activity triggered by Zn but not by Cd. Fig. 1 View largeDownload slide (A and B) Sequence characteristics of AtPCS1 mutants cad1-3 and cad1-6. (A) Genomic situation of AtPCS1. Black and light blue boxes indicate untranslated regions and coding regions, respectively. The sites of a point mutation (cad1-3) and T-DNA insertion (cad1-6) are indicated. (B) Domain organization of AtPCS1. The N-terminal catalytic domain (pfam05023) and the C-terminal Phytochelatin_C domain with unknown functions (DUF1984; pfam09328) are indicated. In cad1-3 there is a nucleotide substitution in exon 4 which results in an amino acid change from tryptophan to cysteine in the N-terminal catalytic domain. No AtPCS1 accumulation is observed in cad1-3 probably due to protein instability (Tennstedt et al. 2009). In cad1-6, the T-DNA insertion disrupts the coding sequence after the codon for Glu409 (Tennstedt et al. 2009), which is located in the latter half of the C-terminal domain. (C–E) Growth assay of the PC-related mutant plants under As(III) stress. (C) Phenotypes of Col-0, cad1-3, cad1-6 and abcc1/2 grown on the agar medium containing 1 or 1.5 μM As(III) for 12 d. Scale bar = 1 cm. Relative primary root length (D) and fresh weight of seedlings (E) of Col-0, cad1-3, cad1-6 and abcc1/2 plants grown on the agar medium containing different concentrations of As(III) for 12 d. Values are shown as a percentage of each control. Data represent means with the SD from two independent experiments (n = 16–24). Means sharing the same letter are not significantly different within each treatment (P < 0.05, Tukey’s HSD). Fig. 1 View largeDownload slide (A and B) Sequence characteristics of AtPCS1 mutants cad1-3 and cad1-6. (A) Genomic situation of AtPCS1. Black and light blue boxes indicate untranslated regions and coding regions, respectively. The sites of a point mutation (cad1-3) and T-DNA insertion (cad1-6) are indicated. (B) Domain organization of AtPCS1. The N-terminal catalytic domain (pfam05023) and the C-terminal Phytochelatin_C domain with unknown functions (DUF1984; pfam09328) are indicated. In cad1-3 there is a nucleotide substitution in exon 4 which results in an amino acid change from tryptophan to cysteine in the N-terminal catalytic domain. No AtPCS1 accumulation is observed in cad1-3 probably due to protein instability (Tennstedt et al. 2009). In cad1-6, the T-DNA insertion disrupts the coding sequence after the codon for Glu409 (Tennstedt et al. 2009), which is located in the latter half of the C-terminal domain. (C–E) Growth assay of the PC-related mutant plants under As(III) stress. (C) Phenotypes of Col-0, cad1-3, cad1-6 and abcc1/2 grown on the agar medium containing 1 or 1.5 μM As(III) for 12 d. Scale bar = 1 cm. Relative primary root length (D) and fresh weight of seedlings (E) of Col-0, cad1-3, cad1-6 and abcc1/2 plants grown on the agar medium containing different concentrations of As(III) for 12 d. Values are shown as a percentage of each control. Data represent means with the SD from two independent experiments (n = 16–24). Means sharing the same letter are not significantly different within each treatment (P < 0.05, Tukey’s HSD). To understand further the element-specific PCS activation mediated by the C-terminal domain, the present study examined the roles of the AtPCS1 C-terminal domain in As(III)-responsive PC synthesis first by phenotyping of cad1-6 and then by in vitro PCS activity assay. We identified two C-terminal regions specifically essential for As-dependent PCS activity and demonstrated significance of the C-terminal domain for plant As tolerance and As movement within plants. Our finding that a particular C-terminal truncation yields an AtPCS1 variant hyperactivated by As(III) may offer a chance to develop a tool useful for better restricting As accumulation in above-ground tissues. Results As(III)-sensitive phenotype of cad1-6 is comparable with that of cad1-3 We first examined the sensitivity of cad1-6 to As(III) stress in comparison with cad1-3 and the PC transporter double mutant abcc1/2 (Song et al. 2010). We employed solid agar plates containing a modified Hoagland medium established to examine toxic metal-sensitive phenotypes with reduced metal dosage compared with Murashige and Skoog (MS) medium (Tennstedt et al. 2009, Fischer et al. 2014). The growth of Col-0, cad1-3, cad1-6 and abcc1/2 was examined under a range of As(III) concentrations (Fig. 1C–E). Root and shoot growth of the three mutants were overall impaired by As(III) treatments, whereas Col-0 grew normally without exhibiting symptoms of toxicity up to a concentration of 1.5 μM As(III). The growth of known As-sensitive mutants cad1-3 and abcc1/2 were similarly inhibited by As(III) treatments (Fig. 1C, D), but the seedling weight was slightly greater in abcc1/2 than in cad1-3 (Fig. 1E), suggesting that our sensitivity assay can detect slight phenotypic differences of As(III) sensitivity. Importantly, cad1-6 showed phenotypes very similar to those of cad1-3 under both moderate (1 μM) and higher (1.5 μM) As(III) exposure (Fig. 1C). Purple-colored leaves were observed in both cad1-3 and cad1-6 exposed to 1.5 μM As(III), but not in Col-0 and abcc1/2 (Fig. 1C). The comparable growth retardation of cad1-3 and cad1-6 exposed to As(III) was evident when root length (Fig. 1D) and seedling fresh weight were measured (Fig. 1E): growth of the two AtPCS1 mutants was similarly inhibited by both moderate (0.75 and 1 μM) and relatively high (1.5 μM) As(III) treatments. Taken together, the results of the growth assays under conditions allowing sensitive detection of toxicity effects in the presence of low metal/metalloid doses demonstrated that there is little difference in As(III) sensitivity between cad1-3 and cad1-6. Distribution of As and essential minerals within plants is altered similarly in cad1-3 and cad1-6 For the second phenotype of the mutants, elemental distribution in plants exposed to As(III) was examined. As and essential element concentrations in the roots and shoots were analyzed and then elemental partitioning between shoots and roots was calculated. The As concentrations in shoots and roots of cad1-3 and cad1-6 were similarly different from those of Col-0 (Fig. 2A, B). Distribution of As to shoots (shoot/root ratio) was significantly and equally higher in cad1-3 and cad1-6 than in Col-0 (Fig. 2C). abcc1/2 also showed increased As distribution to shoots compared with Col-0 (Fig. 2C). Fig. 2 View largeDownload slide Arsenic concentrations in shoots (A) and roots (B), and shoot/root ratios of As (C) in the PC pathway mutants under As(III) stress. Col-0, cad1-3, cad1-6 and abcc1/2 were grown on the control agar medium for 10 d and then transferred to the medium containing 5 μM As(III). After 4 d, plants were harvested and As concentrations in roots and shoots were separately quantified by ICP-OES. Data represent means with the SD from three independent experiments (n = 10). Means sharing the same letter are not significantly different (P < 0.05, Tukey’s HSD). Fig. 2 View largeDownload slide Arsenic concentrations in shoots (A) and roots (B), and shoot/root ratios of As (C) in the PC pathway mutants under As(III) stress. Col-0, cad1-3, cad1-6 and abcc1/2 were grown on the control agar medium for 10 d and then transferred to the medium containing 5 μM As(III). After 4 d, plants were harvested and As concentrations in roots and shoots were separately quantified by ICP-OES. Data represent means with the SD from three independent experiments (n = 10). Means sharing the same letter are not significantly different (P < 0.05, Tukey’s HSD). Among essential metals (Fe, Mn, Cu and Zn), significant alterations of Zn accumulation and distribution were observed in the three tested mutants when treated with As(III) (Fig. 3). Under As(III) treatment, Zn distribution to the shoots of the three mutants dropped down to 30% of that of Col-0, derived from both an increase of Zn accumulation in the roots and a decrease in the shoots of the mutants. Such a difference in the Zn shoot/root ratio between the mutants and Col-0 was not observed under control conditions, although both cad1-3 and cad1-6 showed slightly yet significantly reduced shoot Zn concentrations compared with Col-0. In contrast to Zn, there was no significant difference for tissue concentrations and shoot/root ratios of Fe, Mn and Cu among cad1-3, cad1-6 and Col-0 under both control and As(III) conditions. abcc1/2 showed significantly lower concentrations of Fe and Mn in the roots and shoots under As(III) treatment, compared with Col-0 (Fig. 3). Fig. 3 View largeDownload slide Essential microelement concentrations in shoots and roots, and shoot/root ratios in the PC pathway mutants under normal conditions and As(III)-stressed conditions. Col-0, cad1-3, cad1-6 and abcc1/2 were grown on the control agar medium for 10 d and then transferred to the control medium or medium containing 5 μM As(III). After 4 d, plants were harvested, and element concentrations in roots and shoots were separately quantified by ICP-OES. Data represent means with the SD from two independent experiments (n = 5–6) for control conditions and three independent experiments (n = 7–10). Means sharing the same letter are not significantly different within each treatment (P < 0.05, Tukey’s HSD). Fig. 3 View largeDownload slide Essential microelement concentrations in shoots and roots, and shoot/root ratios in the PC pathway mutants under normal conditions and As(III)-stressed conditions. Col-0, cad1-3, cad1-6 and abcc1/2 were grown on the control agar medium for 10 d and then transferred to the control medium or medium containing 5 μM As(III). After 4 d, plants were harvested, and element concentrations in roots and shoots were separately quantified by ICP-OES. Data represent means with the SD from two independent experiments (n = 5–6) for control conditions and three independent experiments (n = 7–10). Means sharing the same letter are not significantly different within each treatment (P < 0.05, Tukey’s HSD). Cysteine and GSH accumulation is altered similarly in cad1-3 and cad1-6 Cysteine and GSH concentrations in the PC-related mutant plants exposed to As(III) were measured (Fig. 4). abcc1/2 basically showed Col-0-like patterns of cysteine and GSH concentrations in both roots and shoots. On the other hand, cysteine and GSH concentrations of cad1-6 and cad1-3 were significantly, and to a similar degree, elevated in both roots (Fig. 4A, B) and shoots (Fig. 4C, D) compared with Col-0. The difference between the AtPCS1 mutants and Col-0 was evident in roots, with 3-fold higher concentrations in the mutants (Fig. 4A, B). Fig. 4 View largeDownload slide Thiol concentrations in the PC pathway mutants under As(III) stress. Col-0, cad1-3, cad1-6 and abcc1/2 were grown on the control agar medium for 10 d and then transferred to the medium containing 5 μM As(III). After 4 d, plants were harvested and cysteine (A and C) and GSH (B and D) concentrations in roots (A and B) and shoots (C and D) were quantified by HPLC after thiol extraction and derivatization. Data represent means with the SD from at least two independent experiments (n = 6). Means sharing the same letter are not significantly different (P < 0.05, Tukey’s HSD). Fig. 4 View largeDownload slide Thiol concentrations in the PC pathway mutants under As(III) stress. Col-0, cad1-3, cad1-6 and abcc1/2 were grown on the control agar medium for 10 d and then transferred to the medium containing 5 μM As(III). After 4 d, plants were harvested and cysteine (A and C) and GSH (B and D) concentrations in roots (A and B) and shoots (C and D) were quantified by HPLC after thiol extraction and derivatization. Data represent means with the SD from at least two independent experiments (n = 6). Means sharing the same letter are not significantly different (P < 0.05, Tukey’s HSD). Defect of PCS activity in cad1-6 exposed to As(III) The results of phenotyping cad1-6 under As(III) exposure suggested that cad1-6 responds to As(III) stress very similarly to cad1-3. Such cad1-3-like phenotypes of cad1-6 further indicated a possibility that PCS activity of cad1-6 in response to As(III) is mostly impaired. We thus measured PC2 and PC3 concentrations in the plants exposed to As(III) (Fig. 5). In roots, Col-0 accumulated the highest amounts of PC2 and PC3, followed by abcc1/2 (Fig. 5A, B). Under this condition, cad1-3 roots showed trace PC2 and PC3 accumulation. The PC3 concentration in cad1-6 roots was also under the limit of quantification of our HPLC system (Fig. 5B), and only very slight PC2 accumulation was detected in cad1-6 roots (Fig. 5A). In shoots, no detectable PCs were found in either cad1-3 or cad1-6, whereas Col-0 and abcc1/2 accumulated substantial amounts of PC2 and PC3 (Fig. 5C, D). Fig. 5 View largeDownload slide PC concentrations in the PC pathway mutants under As(III) stress. Col-0, cad1-3, cad1-6 and abcc1/2 were grown on the control agar medium for 10 d and then transferred to the medium containing 5 μM As(III). After 4 d, plants were harvested and PC2 (A and C) and PC3 (B and D) concentrations in roots (A and B) and shoots (C and D) were quantified by HPLC after thiol extraction and derivatization. Data represent means with the SD from at least two independent experiments (n = 4–6). Means sharing the same letter are not significantly different (P < 0.05, Tukey’s HSD). Fig. 5 View largeDownload slide PC concentrations in the PC pathway mutants under As(III) stress. Col-0, cad1-3, cad1-6 and abcc1/2 were grown on the control agar medium for 10 d and then transferred to the medium containing 5 μM As(III). After 4 d, plants were harvested and PC2 (A and C) and PC3 (B and D) concentrations in roots (A and B) and shoots (C and D) were quantified by HPLC after thiol extraction and derivatization. Data represent means with the SD from at least two independent experiments (n = 4–6). Means sharing the same letter are not significantly different (P < 0.05, Tukey’s HSD). As(III)-dependent PCS activity of C-terminally truncated AtPCS1 mutants in the fission yeast expression system To examine further the roles of the C-terminal regions of AtPCS1, we analyzed As(III)-dependent PCS activity of the AtPCS1 deletion series using the fission yeast expression system (Kühnlenz et al. 2016). In addition to the wild-type full-length AtPCS1, four different truncated AtPCS1 mutants were examined (Fig. 6A). In a previous study, expression of these mutants in the Schizosaccharomyces pombe PCS knockout strain Δpcs (Clemens et al. 1999) demonstrated differential responses of the mutant proteins to Cd and Zn exposure, and suggested respective protein parts as being required for the PCS activity responding to Cd or Zn (Kühnlenz et al. 2016). We first confirmed the expression of the wild-type AtPCS1 and four truncated variants in the respective cell lines exposed to As(III) (Supplementary Fig. S1). Using this established system, the PCS activity was measured by exposing the cells to As(III). PC2 and PC3 were substantially synthesized in the cells with the wild-type AtPCS1, whereas both PC2 and PC3 levels in the cells with the empty vector were under the limit of quantification of our analytical system (Fig. 6B). Under this condition, cells expressing the shortest truncated mutant Δ373–485 did not accumulate PC2 and PC3 above the quantification levels like cells carrying the empty vector (Fig. 6B). Cells expressing longer mutants Δ460–485 and Δ471–485 showed statistically similar PC2 and PC3 accumulation: the mutants accumulated approximately 25–50% of PC2 and 60% of PC3 compared with the wild-type AtPCS1-expressing cells. Interestingly, expression of the longest version, Δ476–485, resulted in cells accumulating higher levels of PC2 and PC3 compared with the cells with wild-type AtPCS1. Fig. 6 View largeDownload slide PCS activity assay of AtPCS1 deletion mutants of the C-terminal domain in response to As(III). (A) Schematic of full-length AtPCS1 and a series of C-terminally truncated AtPCS1s expressed in the S. pombe PCS knockout strain Δpcs. (B) PC concentrations in the S. pombe cells exposed to 10 μM As(III) for 4 h. PC2 and PC3 were quantified by HPLC after thiol extraction from the harvested cells and derivatization. Data represent means with the SD from two independent experiments (n = 4). Means sharing the same letter are not significantly different (P < 0.05, Tukey’s HSD). (C) Growth of the S. pombe Δpcs cells harboring an empty vector pSGP72 or expressing AtPCS1 or truncated variants exposed to As(III). Data represent means with the SD from two independent experiments (n = 8). Means sharing the same letter are not significantly different within each treatment (P < 0.05, Tukey’s HSD). Fig. 6 View largeDownload slide PCS activity assay of AtPCS1 deletion mutants of the C-terminal domain in response to As(III). (A) Schematic of full-length AtPCS1 and a series of C-terminally truncated AtPCS1s expressed in the S. pombe PCS knockout strain Δpcs. (B) PC concentrations in the S. pombe cells exposed to 10 μM As(III) for 4 h. PC2 and PC3 were quantified by HPLC after thiol extraction from the harvested cells and derivatization. Data represent means with the SD from two independent experiments (n = 4). Means sharing the same letter are not significantly different (P < 0.05, Tukey’s HSD). (C) Growth of the S. pombe Δpcs cells harboring an empty vector pSGP72 or expressing AtPCS1 or truncated variants exposed to As(III). Data represent means with the SD from two independent experiments (n = 8). Means sharing the same letter are not significantly different within each treatment (P < 0.05, Tukey’s HSD). We then examined growth of the cells expressing AtPCS1 or the truncated variants under different concentrations of As(III) (Fig. 6C). The cells expressing the full-length AtPCS1 showed about 2-fold better growth than control cells with an empty vector under both 25 and 50 μM As(III). Under these conditions, growth of the cells expressing the shortest truncated mutant Δ373–485 was statistically equal to that of empty vector controls, whereas the cells expressing Δ460–485 or Δ471–485 showed intermediate tolerance to As(III). The cells expressing Δ476–485 represented slightly yet significantly better growth under both As(III) conditions compared with the cells expressing the wild-type AtPCS1 (Fig. 6C). Enhanced PCS activity of Δ476–485 recombinant protein in response to As(III) To examine further the As(III)-dependent PCS activity of Δ476–485 by the in vitro assay (Fischer et al. 2014), we purified the recombinant proteins of Δ476–485 and the wild-type AtPCS1 (Supplementary Fig. S2A). As(III)-dependent PCS activities of the recombinant proteins were assessed relative to Cd-dependent activities of the respective proteins as previously described (Oven et al. 2002, Fischer et al. 2014). Relative As(III)-dependent PCS activity of the wild-type protein in the presence of 100 μM As(III) was 19% of the activity in the presence of 100 μM Cd (Supplementary Fig. S2B), which was very similar to the reported data for purified AtPCS1 (Oven et al. 2002). Compared with the wild-type AtPCS1, Δ476–485 clearly showed much higher As(III)-dependent PCS activity relative to Cd-dependent activity. This difference was also observed when 10 μM As(III) or Cd were added to the assay mixtures. Discussion Due to human activities or geochemical factors, phytoavailability of several elements such as Cd, As, Zn and aluminum (Al) in soils can reach levels toxic to plants. Since plants are sessile, they have to cope with such elemental toxicity in the rhizosphere. Molecular mechanisms underlying plant tolerance against soil-derived elemental stress have been revealed for several cases, and many of the identified proteins are generally associated with a single elemental stress. For example, AtMTP1, a tonoplast Zn transporter, confers the excess Zn tolerance to A. thaliana (Kobae et al. 2004, Desbrosses-Fonrouge et al. 2005); however, AtMTP1 is probably not involved in Cd, nickel (Ni) and manganese (Mn) tolerance (Kobae et al. 2004, Weber et al. 2013). In contrast, the PC/PCS system is associated with detoxification of a wide range of elemental toxicity. Analyses of cad1-3 and abcc1/2 suggested the significant role of PC synthesis and subsequent vacuolar compartmentation in detoxifying toxic inorganic ions such as Cd, Hg and As (Howden et al. 1995, Ha et al. 1999, Song et al. 2010, Park et al. 2011). AtPCS1 also plays an important role in detoxification of Pb and excess Zn (Tennstedt et al. 2009, Fischer et al. 2014). Various other elements including Cu, silver (Ag) and antimony (Sb) are also able to induce PC synthesis in vitro, but iron (Fe) and Mn are not (Grill et al. 1987, Cazale and Clemens 2001, Kühnlenz et al. 2014). Such responsiveness of PCS to a relatively wide range of elements raised the question as to how PCS is activated by these different elements. It was suggested that the AtPCS1 C-terminal region is involved in AtPCS1 activation by different metals such as Cd and Zn (Ruotolo et al. 2004, Tennstedt et al. 2009, Kühnlenz et al. 2016). However, mechanisms of As-dependent AtPCS1 activation have been little examined, whereas the physiological functions of PCS/PC have been well demonstrated in regard to As exposure. Moreover, understanding mechanisms of As-dependent PCS activation could be potentially applied for molecular breeding of PCS variants hyperactivated by As(III). For Cd, a previous study conducted site-directed mutagenesis of AtPCS1 and identified variants which enhance Cd tolerance (Cahoon et al. 2015). The present study aimed to understand roles of the AtPCS1 C-terminal domain in As(III) responses and search for variants with enhanced As(III)-dependent PCS activity. We first characterized phenotypes of cad1-6 in comparison with the null mutant cad1-3 under As(III) stress conditions. cad1-6 has a T-DNA insertion in exon 8 of AtPCS1, disrupting the coding sequence after the codon for Glu409 (Fig. 1A, B) (Tennstedt et al. 2009). Due to the T-DNA insertion, cad1-6 is suggested to express a C-terminally truncated AtPCS1 (Tennstedt et al. 2009, Kühnlenz et al. 2016). To examine the As(III) sensitivity of cad1-6, we employed the growth assay which was able to detect slight differences of growth between cad1-3 and abcc1/2. Under all As(III) treatments tested, the phenotypes of cad1-6 closely resembled those of cad1-3: there were no significant differences in root length (Fig. 1D) and plant fresh weight between the AtPCS1 mutants (Fig. 1E). Both AtPCS1 mutants accumulated anthocyanin in shoots under 1.5 μM As(III) (Fig. 1C), whereas abcc1/2 did not show such pigment accumulation. The cad1-3 like As sensitivity of cad1-6 suggested that PCS activity in response to As(III) is diminished in cad1-6 to the level of cad1-3. Similarly, As accumulation (Fig. 2) as well as cysteine and GSH concentrations (Fig. 4) in cad1-6 were also equal to those of cad1-3 under As(III) treatment, further supporting the hypothesis. Concerning As accumulation, the distribution of As to the shoot was significantly higher in cad1-6 and cad1-3 compared with the wild type Col-0 when exposed to As(III) (Fig. 2C). A previous study also reported increased As translocation to shoots of cad1-3 exposed to As(V) and suggested that AtPCS1-mediated PC synthesis in response to As facilitates vacuolar compartmentation of PC–As complexes in root cells and thus retains As in the roots (Liu et al. 2010). Increased As distribution in cad1-6 shoots comparable with cad1-3 may explain the stronger toxicity symptoms of cad1-6 and cad1-3 shoots relative to abcc1/2 (Fig. 1C), and suggests a defect of PC synthesis and PC–As complex formation in cad1-6 exposed to As(III). In line with the hypothesis, in cad1-6 exposed to As(III), only trace PCs were detected, as found for cad1-3, both in shoots and in roots (Fig. 5), demonstrating that As(III)-dependent PCS activity is impaired in cad1-6 to the same extent as in the null mutant cad1-3. Likewise, abolished AtPCS1 activity of cad1-6 in response to As(III) is reflected in increased GSH and cysteine accumulation (Fig. 4). Similar trace PC accumulation was observed when the AtPCS1 mutant plants were exposed to excess Zn, whereas Cd exposure led to substantial PC2 accumulation in cad1-6 roots (Tennstedt et al. 2009). The different PC accumulation between cad1-6 and cad1-3 exposed to Zn or Cd suggested that C-terminally truncated AtPCS1 expression expected in cad1-6 can be activated by Cd but not by Zn (Tennstedt et al. 2009). Indeed, in vitro experiments and heterologous expression suggested that the stretch between His460 and Arg470 of AtPCS1 is as an essential C-terminal region for activation by Zn, but for Cd-triggered activation the regions between Glu283 and Asp372 as well as the N-terminal domain are crucial (Ruotolo et al. 2004, Romanyuk et al. 2006, Kühnlenz et al. 2016). The cad1-3-like phenotypes of cad1-6 under As(III) stress demonstrated in this study (Figs. 1–5) indicate a significant role for the AtPCS1 C-terminal domain in As-dependent PCS activity as is the case for Zn excess-dependent activation. To examine this hypothesis further, a series of AtPCS1 C-terminal deletion mutants (Kühnlenz et al. 2016; Fig. 6A) was expressed in the fission yeast expression system (Supplementary Fig. S1), and their PCS activities in response to As(III) were examined. The C-terminal region spanning Asp373 to Leu459 was suggested to be crucial for the activation by As(III) (Fig. 7), since the shortest mutant Δ373–485 did not show any PC synthesis activity above the background level of the empty vector control, while a longer mutant Δ460–485 was able to synthesize an appreciable amount of PCs (Fig. 6B). The stretch between His460 and Arg470 essential for activation by Zn (Kühnlenz et al. 2016) is less likely to be important for activation by As(III), because there was no significant effect of adding the stretch from His460 to Arg470 when comparing the activities of Δ460–485 and Δ471–485. Cys471 to Lys475 was suggested as the second crucial amino acid stretch for activation by As(III) (Fig. 7) based on the remarkable increase of PC accumulation in Δ476–485 compared with the shorter mutants. Considering that the As(III)-dependent PC accumulation in Δ476–485 exceeded even that of the wild-type AtPCS1 (Fig. 6B; Supplementary Fig. S2) which conferred additional As(III) tolerance to the cells (Fig. 6C), it is also possible that the amino acid residues after Glu476 may negatively affect the PCS activation by As(III). Fig. 7 View largeDownload slide Partial amino acid sequences of the AtPCS1 C-terminal domain. The two regions suggested to be important for As(III)-dependent activation in the present study are indicated (Asp373 to Leu459 and Cys471 to Lys475). The regions for Cd- or Zn-dependent activation are also indicated based on the results of Kühnlenz et al. (2016). Fig. 7 View largeDownload slide Partial amino acid sequences of the AtPCS1 C-terminal domain. The two regions suggested to be important for As(III)-dependent activation in the present study are indicated (Asp373 to Leu459 and Cys471 to Lys475). The regions for Cd- or Zn-dependent activation are also indicated based on the results of Kühnlenz et al. (2016). Overall, the deletion analysis of the AtPCS1 C-terminal domain suggests at least two regions important for AtPCS1 activation by As(III): Asp373 to Leu459 and Cys471 to Lys475 (Fig. 7). The former region contains Glu409, after which the coding sequence is disrupted by the T-DNA insertion in cad1-6 (Fig. 1B) (Tennstedt et al. 2009). Taken together with the abolished PCS activity in cad1-6 exposed to As(III) (Fig. 5), it may be possible to narrow down the candidate region to the sequence from after Glu409 to Leu459. Compared with the suggested regions in the C-terminal domain important for AtPCS1 activation by Cd and excess Zn (Ruotolo et al. 2004, Kühnlenz et al. 2016), we would suggest that the two regions for As-dependent activation are unique (Fig. 7). Through the phenotyping of the PC-related mutant plants exposed to As(III), we observed the drastic reduction of Zn accumulation in the AtPCS1 mutants exposed to As(III) (Fig. 3). Such a reduction of the Zn shoot/root ratio was not observed under control conditions; however, cad1-3 and cad1-6 showed slight yet significant reduction of Zn concentrations in shoots. A previous study suggested that AtPCS1 controls Zn delivery from roots to shoots under normal Zn conditions (Kühnlenz et al. 2016). Our results support the idea and further suggest that the contribution of AtPCS1-dependent Zn delivery to shoots is more evident under As(III) stress. Enhanced PC production triggered by As(III) in root cells may facilitate PC–Zn complex formation for translocation to shoots and to maintain Zn levels in shoots. Alternatively, other PC-independent Zn translocation pathways are affected in As(III)-exposed plants, which would increase the importance of PC-dependent Zn mobility. The same Zn reduction was observed for abcc1/2 mutant plants, confirming that alteration of PC metabolism and/or intracellular transport may also affect Zn homeostasis. It should be noted that abcc1/2 also showed reduced accumulation of Fe and Mn in the roots and shoots (Fig. 3). In conclusion, we suggest the that the two unique stretches of the AtPCS1 C-terminal region are crucial for As(III)-dependent activation. The analyses of cad1-6 suggest that the C-terminal regions are crucial for in planta PC synthesis in response to As(III) stress and eventually for As(III) detoxification and As mobility within plants. It should be further investigated which amino acid residues are the keys for As(III)-dependent AtPCS1 activation in vitro and in planta as well. Cysteine is established as the major amino acid binding to As(III) (Zhou et al. 2011, Shen et al. 2013). Hydrophilic amino acids such as arginine and aspartate are also suggested as important residues to form As-binding sites together with cysteine (Bennett et al. 2001, Martin et al. 2001). The two identified regions of AtPCS1 crucial for As(III)-dependent activation include cysteine, and the longer stretch Asp373 to Leu459 contains arginine and aspartate residues. Such amino acid residues within the regions would be primary candidates mediating As(III)-dependent AtPCS1 activation. It should also be noted that the C-terminal regions are highly conserved among higher plant PCSs (Kühnlenz et al. 2016). Another interesting finding is that the Δ476–485 variant which lacks the C-terminal 10 amino acid residues shows enhanced PCS activity compared with the wild-type protein in response to As(III). Since PCS contributes to restricting As mobility in plants (Liu et al. 2010, Hayashi et al. 2017, Uraguchi et al. 2017), this variant could potentially be used as a tool to decrease As distribution to seeds or grains. Materials and Methods Plant materials and growth conditions Arabidopsis thaliana wild type (Col-0), the AtPCS1 null mutant cad1-3 (Howden et al. 1995), the AtPCS1 T-DNA insertion line cad1-6 (Tennstedt et al. 2009) and the AtABCC1 and AtABCC2 double knockout mutant line abcc1/2 (Song et al. 2010) were used in this study. For As(III) sensitivity assay, agar plates containing 1/10th modified Hoagland medium were used for plant cultivation (Tennstedt et al. 2009, Fischer et al. 2014) [100 μM (NH4)2HPO4, 200 μM MgSO4, 280 μM Ca(NO3)2, 600 μM KNO3, 5 μM Fe-HBED, 1% (w/v) sucrose, 5 mM MES, 1% (w/v) purified agar (Nacalai Tesque), pH 5.7]. When cultivating plants for elemental and PC analyses, the following microelements were additionally supplemented to the medium (4.63 μM H3BO3, 32 nM CuSO4, 915 nM MnCl2, 77 nM ZnSO4, 11 nM MoO3) (Kühnlenz et al. 2014). Arabidopsis seeds were surface sterilized and sown on agar plates. After 2 d stratification at 4°C, plants were grown vertically in a growth chamber (16 h light/8 h dark, 22°C) as described elsewhere. As(III) sensitivity assay To examine the sensitivity of Arabidopsis to As(III), plants were grown for 12 d on agar plates containing different concentrations of As(III) as NaAsO2 (0.75, 1 and 1.5 μM). The plates without addition of As(III) served as controls. Plant growth was assessed by primary root length and seedling fresh weight measurements at the end of the cultivation. Elemental analysis For elemental analyses of plant samples, plants were grown on the control plates (solidified with 1.5% agar) for 10 d. Uniformly grown seedlings were then transferred to control plates or plates containing 5 μM As (III) and incubated for an additional 4 d. Roots and shoots from each plate (normally 15 seedlings per genotype) were separately pooled as a single sample. Shoot samples were washed with MilliQ water twice. Root samples were subjected to sequential washing procedures: roots were desorbed for 10 min each in ice-cold MilliQ water, 20 mM CaCl2 (twice), 10 mM EDTA (pH 5.7) and MilliQ water. Harvested roots and shoots were dried at 50°C before acid digestion. Dried plant samples were wet-digested with HNO3. Elemental concentrations in the samples were quantified by inductively coupled plasma-optical emission spectroscopy (ICP-OES; iCAP7400Duo, Thermo-Fisher Scientific). PC analysis of plant samples For PC analyses of plant samples, plants were grown on the control plates (solidified with 1% agar) for 10 d. Uniformly grown seedlings were then transferred to plates containing 5 μM As(III) and incubated for an additional 4 d. Roots and shoots from each plate (normally 15 seedlings per genotype) were separately pooled as a single sample. The plant samples were frozen in liquid nitrogen after fresh weight measurement. Homogenously ground material was used for thiol extraction. Thiols were extracted and derivatized as described, with a slight modification (Kühnlenz et al. 2014). A 10 mg aliquot of plant material was extracted with 30 μl of 0.1% (v/v) trifluoroacetic acid (TFA) containing 6.3 mM diethylenetriaminepentaacetic acid (DTPA). Thiol derivatives were analyzed by HPLC equipped with a fluorescence detector (Nishida et al. 2016). Quantification of thiol derivatives was performed via standards of cysteine, GSH, PC2 and PC3 after normalization to the internal N-acetylcysteine (NAC) standard. Standards of PC2 and PC3 were synthesized by Bonac Corporation (http://www.bonac.com). Heterologous expression of AtPCS1 and the truncated variants in fission yeast For the in vivo testing of PCS variants, the fission yeast (S. pombe) PCS knockout strain Δpcs (Clemens et al. 1999) heterologously expressing HA-tagged AtPCS1 or a series of C-terminal deletion mutants were used (Kühnlenz et al. 2016). Cells carrying the empty vector pSGP72 served as negative control. The assay was conducted as described previously, with some modifications (Kühnlenz et al. 2016). Yeast cultivation was carried out at 30°C in Edinburgh’s minimal medium (EMM) supplemented with 1 μM thiamine. Pre-cultured cells were inoculated to an OD600 = 0.1 in EMM supplemented with 1 μM thiamine and grown overnight. Then cells were inoculated at an OD600 = 0.5 in EMM supplemented with 1 μM thiamine and 10 μM As(III). After 4 h incubation, the OD600 was measured for each culture to assess cell numbers. Cells were then harvested and frozen in liquid N2 prior to protein and PC extraction. For detection of the HA-tagged AtPCS1 and truncated proteins, protein extracts were prepared from the harvested cells as described, with minor modifications (Matsuo et al. 2006), and were subjected to Western blotting using the anti-HA tag antibody (MBL, M180-3) as the primary antibody. Horseradish peroxidase-conjugated goat anti-mouse IgG (GE Healthcare, NA931V) was used as the secondary antibody and Chemi Lumi One L (Nacalai Tesque) was used for detection. For PC quantification, the extraction buffer [0.1% (v/v) TFA containing 6.3 mM DTPA] was used for thiol extraction from the harvested cells. Thiol derivatization and quantification by HPLC were performed as described elsewhere. For growth assay, pre-cultured cells were prepared using EMM supplemented with 1 μM thiamine. Then cells were inoculated at an OD600 = 0.1 in EMM supplemented with 1 μM thiamine and two different concentrations of As(III) (25 and 50 μM). Growth of the cells was monitored by measuring the OD600 for 24 h. Recombinant protein production and PCS activity assay Sequences coding for the full-length AtPCS1 protein and the truncated variant Δ476–485 were amplified from cDNA using the primers listed in Supplementary Table S1 and cloned into the expression vector pET19TEV in-frame with an N-terminal 6×His tag. The expression plasmids were transformed into Escherichia coli Rosetta2(DE3)pLysS cells. Expression was induced at an OD600 of about 0.6 by addition of 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Cells were grown for 14 h at 21°C and 150 r.p.m., after which they were harvested (25 min, 4°C, 6,160×g). Soluble His-tagged recombinant protein was purified using an Ni-NTA matrix (Qiagen) as previously described (Fischer et al. 2014). Protein concentrations were determined using Roti®-Quant (Carl Roth) according to Bradford, and estimated based on a bovine serum albumin (BSA) dilution series following SDS–PAGE. PCS activity was assessed by adding heterologously expressed protein in 20 μl of storage buffer [50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 7.0, 25% (v/v) glycerol] to 80 μl of activity buffer [50 mM HEPES pH 7.0, 12.5 mM GSH, 10% (v/v) glycerol]. Enzymes were activated by addition of Cd(II) or As(III) at the indicated concentrations. The reaction was allowed to proceed for up to 60 min at 35°C and stopped by addition of 1.1 μl of 10% (v/v) TFA. Thiols were extracted, monobromobimane-derivatized and PC2 was quantified by HPLC as described (Kühnlenz et al. 2014). Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the Japan Society for the Promotion of Science [grant No. 16K14873 to S.U.] and the Deutsche Forschungsgemeinschaft [CL 152/7-2 to S.C.]. Acknowledgments We thank Youngsook Lee (POSTECH) and Enrico Martinoia (University of Zurich) for providing abcc1/2 seeds, and Silke Matros for excellent technical assistance. Standards of PCs were kindly provided by Shin-ichi Nakamura (Tokyo University of Agriculture). Disclosures The authors have no conflicts of interest to declare. References Bennett M.S., Guan Z., Laurberg M., Su X.D. ( 2001) Bacillus subtilis arsenate reductase is structurally and functionally similar to low molecular weight protein tyrosine phosphatases. Proc. Natl. Acad. Sci. USA  98: 13577– 13582. 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Plant  8: 722– 733. Google Scholar CrossRef Search ADS PubMed  Zhao L., Chen S., Jia L., Shu S., Zhu P., Liu Y. ( 2012) Selectivity of arsenite interaction with zinc finger proteins. Metallomics  4: 988– 994. Google Scholar CrossRef Search ADS PubMed  Zhou X., Sun X., Cooper K.L., Wang F., Liu K.J., Hudson L.G. ( 2011) Arsenite interacts selectively with zinc finger proteins containing C3H1 or C4 motifs. J. Biol. Chem.  286, 22855– 22863. Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations DTPA diethylenetriaminepentaacetic acid EMM Edinburgh’s minimal medium GSH glutathione ICP-OES inductively coupled plasma-optical emission spectroscopy PC phytochelatin PCS phytochelatin synthase TFA trifluoroacetic acid © The Author(s) 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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Plant and Cell PhysiologyOxford University Press

Published: Mar 1, 2018

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