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OsHAC4 is critical for arsenate tolerance and regulates arsenic accumulation in rice Jiming Xu1*, Shulin Shi2*, Lei Wang1, Zhong Tang2, Tingting Lv1, Xinlu Zhu1, Xiaomeng Ding1, Yifeng Wang1, Fang-Jie Zhao2,3 and Zhongchang Wu1 1
State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China; 2State Key Laboratory of Crop Genetics and Germplasm
Enhancement, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, China; 3Rothamsted Research, Harpenden Hertfordshire, AL5 2JQ, UK
Summary Authors for correspondence: Zhongchang Wu Tel: +86 571 88981395 Email:
[email protected] Fang-Jie Zhao Tel: +86 25 8439 6509 Email:
[email protected] Received: 23 January 2017 Accepted: 12 March 2017
New Phytologist (2017) 215: 1090–1101 doi: 10.1111/nph.14572
Key words: arsenate, arsenate reductase, arsenic (As), arsenic accumulation, arsenite, detoxification, rice (Oryza sativa).
Soil contamination with arsenic (As) can cause phytotoxicity and elevated As accumulation in rice grain. Here, we used a forward genetics approach to investigate the mechanism of arsenate (As(V)) tolerance and accumulation in rice. A rice mutant hypersensitive to As(V), but not to As(III), was isolated. Genomic resequencing and complementation tests were used to identify the causal gene. The function of the gene, its expression pattern and subcellular localization were characterized. OsHAC4 is the causal gene for the As(V)-hypersensitive phenotype. The gene encodes a rhodanase-like protein that shows As(V) reductase activity when expressed in Escherichia coli. OsHAC4 was highly expressed in roots and was induced by As(V). In OsHAC4pro-GUS transgenic plants, the gene was expressed exclusively in the root epidermis and exodermis. OsHAC4-eGFP was localized in the cytoplasm and the nucleus. Mutation in OsHAC4 resulted in decreased As(V) reduction in roots, decreased As(III) efflux to the external medium and markedly increased As accumulation in rice shoots. Overexpression of OsHAC4 increased As (V) tolerance and decreased As accumulation in rice plants. OsHAC4 is an As(V) reductase that is critical for As(V) detoxification and for the control of As accumulation in rice. As(V) reduction, followed by As(III) efflux, is an important mechanism of As(V) detoxification.
Introduction Arsenic (As) contamination is widespread in paddy soils in south and southeast Asia as a result of mining and irrigation with Ascontaminated groundwater (Brammer, 2009; Williams et al., 2009; Zhao et al., 2015). The build-up of As in paddy soils can lead to phytotoxicity and substantial yield losses in rice, threatening agricultural sustainability in the affected areas (Panaullah et al., 2009). Moreover, As contamination in paddy soils can increase As accumulation in rice grain, thus posing a risk to food safety and human health (Meharg & Rahman, 2003; Zhu et al., 2008). It is known that rice is a major dietary source of inorganic As, a class one carcinogen, for populations consuming rice as the staple food (Kile et al., 2007; Meharg et al., 2009; Li et al., 2011). Therefore, it is important to understand the mechanisms of As tolerance and accumulation in rice in order to develop strategies to minimize the risk of As contamination in soil. Plants take up different chemical species of As inadvertently via transporters for essential or beneficial elements as a result of imperfect substrate selectivity. For example, arsenate (As(V)), the predominant As species in aerobic soil, is taken up by plant roots *These authors contributed equally to this work.
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via phosphate transporters (Shin et al., 2004; Catarecha et al., 2007), such as OsPT1 and OsPT8 in rice (Wu et al., 2011; Kamiya et al., 2013; Wang et al., 2016). By contrast, arsenite (As (III)), the predominant As species in anaerobic soil, is transported across the plasma membranes via some aquaporin channels (Bienert et al., 2008; Isayenkov & Maathuis, 2008; Kamiya et al., 2009; Zhao et al., 2009; Xu et al., 2015). In the case of rice, the silicon transporters Lsi1 (OsNIP2;1) and Lsi2 are the main uptake pathways for As(III) (Ma et al., 2008). Lsi1 is an aquaporin channel responsible for the entry of As(III) into the root cells, whereas Lsi2 is an efflux transporter mediating the efflux of As(III) towards the stele for xylem loading (Ma et al., 2008). Methylated As species can also be taken up via Lsi1 in rice roots (Li et al., 2009). Arsenic is toxic to plants, although the mode of toxicity differs among different As species (Zhao et al., 2009; Finnegan & Chen, 2012). As(V) interferes with phosphate (Pi) metabolism by participating in phosphorylation reactions, forming arsenate esters that are much less stable than phosphate esters (Byers et al., 1979; Finnegan & Chen, 2012). The unstable arsenate esters hydrolyse quickly, leading to the uncoupling of phosphorylation and disruption of glucose and energy metabolism (Byers et al., 1979). By contrast, As(III) binds to reduced cysteine residues in proteins, Ó 2017 The Authors New Phytologist Ó 2017 New Phytologist Trust
New Phytologist affecting their structures or catalytic functions (Shen et al., 2013). Exposure to As(V) or As(III) also induces the generation of reactive oxygen species (ROS) which can damage macromolecules and cell membranes (Finnegan & Chen, 2012). Plants employ different mechanisms to detoxify As(V) and As(III). As(III) is detoxified primarily by complexation with thiol-rich peptides, such as glutathione (GSH) and phytochelatins (PCs) (Raab et al., 2005; Liu et al., 2010). Mutants of GSH and PC synthesis are hypersensitive to As (Ha et al., 1999; Liu et al., 2010; Tang et al., 2016; Yang et al., 2016). As(III)–PC complexes are transported into the vacuoles via ABCC transporters, a process that is also important for As(III) detoxification (Song et al., 2010, 2014). For the detoxification of As(V), As(V) must be reduced to As(III) first, which can then be detoxified via thiol complexation and subsequent vacuolar sequestration. Following As(V) uptake by plant roots, a large proportion (typically 60–80%) of the As taken up is extruded to the external medium as As(III) (Xu et al., 2007; Liu et al., 2010; Zhao et al., 2010a). This As(III) efflux lessens the cellular burden of As and is likely to represent an important component of As(V) detoxification in plants, as has been shown to be the case in microorganisms (Bhattacharjee & Rosen, 2007). Recently, Chen et al. (2016) have proposed an alternative mechanism of As(V) detoxification in microorganisms via the formation of 1-arseno-3-phosphoglycerate and the subsequent efflux of this unstable organoarsenical compound out of the cell. It is not known whether a similar mechanism also exists in plant roots. It is clear that As(V) reduction is a key step of detoxification in plants. Following As(V) uptake, As(V) is readily reduced to As (III), suggesting a high capacity for As(V) reduction (Pickering et al., 2000; Dhankher et al., 2006; Xu et al., 2007; Zhao et al., 2009). Earlier studies have suggested that plant ACR2 proteins, which are homologues of the yeast As(V) reductase ACR2, may be responsible for As(V) reduction (Dhankher et al., 2006; Duan et al., 2007). However, more recent studies have cast doubt on the in planta role of ACR2 in As(V) reduction (Liu et al., 2012; Chao et al., 2014). Two recent studies have independently identified a new As(V) reductase in Arabidopsis thaliana, named HAC1 (High As Content 1) (Chao et al., 2014) or ATQ1 (Arsenic Tolerance QTL 1) (Sanchez-Bermejo et al., 2014). HAC1/ATQ1 reduces As(V) to As(III) when the gene is expressed heterologously in E. coli. Weak or null alleles of HAC1/ATQ1 in Arabidopsis accessions are associated with decreased tolerance to As (V) (Chao et al., 2014; Sanchez-Bermejo et al., 2014) and massively increased As accumulation in shoots (Chao et al., 2014). The hac1 null mutants lose most of the As(III) efflux capacity observed in wild-type (WT) plants, which, in turns, leads to the hyperaccumulation of As in above-ground tissues (Chao et al., 2014). Using a reverse genetics approach, Shi et al. (2016) have shown that two orthologues of HAC1 in rice, OsHAC1;1 and OsHAC1;2, also function as As(V) reductases and play a role in the control of As accumulation in rice. There are > 10 AtHAC1-like genes in the rice genome (Shi et al., 2016). Except for OsHAC1;1 and OsHAC1;2, the functions of other AtHAC1-like genes have not been characterized. In the present study, we isolated an As(V)-hypersensitive mutant of rice Ó 2017 The Authors New Phytologist Ó 2017 New Phytologist Trust
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and identified OsHAC4 (Loc_Os02g06290) as the causal gene for the mutant phenotype. Our results show that OsHAC4 encodes an As(V) reductase that is critical for As(V) tolerance and plays an important role in the control of As accumulation in rice plants.
Materials and Methods Plant materials and growth conditions The Oshac4/Osphf1-7 mutant was isolated from an ethyl methanesulfonate (EMS)-generated rice (Oryza sativa japonica, cv HJ2 with a mutation in OsPHF1) mutant library, based on a root elongation assay in a nutrient solution containing 20 lM Na3AsO4 (Yang et al., 2016). After germination, seedlings were grown hydroponically in a nutrient solution designed for rice culture (Yoshida et al., 1976) with Fe being supplied as 40 lM NaFe(III)EDTA. The solution was adjusted to pH 5.5 using 1 M HCl or 1 M NaOH before use, and renewed every 3 d. Rice plants were grown in a growth chamber at 30°C : 22°C (12 h : 12 h, day : night) and c. 60% relative humidity. Arsenic treatments were started by the addition of As(V) (Na3AsO4) or As(III) (NaAsO2) to the nutrient solution at target concentrations. Cloning of OsHAC4 and construction of complementation To clone the causal gene responsible for the mutant phenotype, the As(V)-hypersensitive mutant (in the Osphf1-7 background) was backcrossed with its WT Osphf1-7. The F2 progeny plants which showed hypersensitivity to 20 lM Na3AsO4 were selected for gene cloning using the MUTMAP method (Abe et al., 2012). Briefly, DNA was extracted from 25 F2 As(V)-hypersensitive individuals and mixed in an equal ratio. A 5 lg mixed DNA library was prepared for Illumina sequencing. Genomic resequencing was performed with the DNA library to aim for a mean coverage of 309 using a read length of 2 9 100 bp on the Illumina HiSeq2500 sequencer, as described previously (Yang et al., 2016). Based on the data from genomic resequencing, a cleaved amplified polymorphic sequence (CAPS) marker was developed to confirm the mutated site of the candidate gene. The PCR products were digested with BslI. The primers used are listed in Supporting Information Table S1. After the mutant gene had been cloned and confirmed, the Oshac4/Osphf1-7 mutant was backcrossed to HJ2 to generate the single mutant Oshac4 in the HJ2 background. For complementation of the Oshac4 mutant, the full-length coding sequence of OsHAC4 (402 bp) fused in-frame to the 50 terminus of Flag was cloned into the binary vector and driven by its native promoter (c. 3.1 kb). The binary vector was introduced into Oshac4 mutants by Agrobacterium-mediated transformation (Chen et al., 2003). Functional complementation of OsHAC4 in E. coli For the expression of OsHAC4 in E. coli, the full-length coding sequences of OsHAC4, as well as a positive control, OsHAC1;1 New Phytologist (2017) 215: 1090–1101 www.newphytologist.com
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(Shi et al., 2016), were amplified using gene-specific primers named OsHAC4-pCold (Table S1). The fragments were cloned into the prokaryotic expression vector pCold-I and verified by sequencing. The vector was transformed into the E. coli DarsC mutant WC3110 and its WT W3110 for complementation. Bacterial strains were cultured at 37°C overnight. All cultured strains were diluted to an optical density at 600 nm (OD600 nm) = 1.0 and 1.0 ml was inoculated into 100 ml of Luria–Bertani (LB) liquid medium containing 1 mM isopropyl b-D-1-thiogalactopyranoside and 1 mM As(V). Cells were cultured at 16°C. The cell density was measured at OD600 nm using a spectrophotometer at different time points. To determine the production of As(III), the overnight cultured strains of the DarsC mutant (WC3110) with pCold-I empty vector, pCold-IOsHAC4 or pCold-I-OsHAC1;1 were diluted to OD600 nm = 1.0, and 0.5 ml was inoculated into 50 ml of LB liquid medium containing 1 mM isopropyl b-D-1-thiogalactopyranoside and 10 lM As(V). Cells were cultured at 16°C. The LB medium was collected at 72 h and filtered through a 0.22-lm membrane filter before As speciation analysis using high-performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS). Generation of OsHAC4 overexpression lines To generate OsHAC4 overexpression lines, the full-length coding sequence of OsHAC4 (402 bp) was cloned and digested with KpnI and XbaI, and ligated to the 35S-pCAMBIA1301 vector (Zhou et al., 2008). The verified vectors were used for the generation of transgenic plants of OsHAC4 in the cv. HJ2 background. The primers are listed in Table S1. RNA extraction and transcriptional analysis by quantitative real-time PCR Total RNAs were isolated using an RNA extraction kit (NucleoSpin RNA Plant, Macherey-Nagel, D€ uren, Germany). A quantitative real-time polymerase chain reaction (qRT-PCR) was conducted as described previously (Chen et al., 2011). The primers for quantitative RT-PCR analyses are listed in Table S1. b-Glucuronidase (GUS) histochemical analysis To identify the tissue localization of OsHAC4 expression, OsHAC4pro-GUS transgenic plants were generated using HJ2 genomic DNA fragments containing the c. 3.1-kb promoter sequence of OsHAC4. The promoter sequence was amplified and inserted into the binary vector GUS-pBI101.3 between the KpnI and BamHI sites. GUS analysis was performed as described previously (Xu et al., 2013). The primers are listed in Table S1. Subcellular localization analysis For analysis of the subcellular localization of OsHAC4, the full-length coding sequences (excluding the stop codons) of OsHAC4 (399 bp) were amplified and inserted into the New Phytologist (2017) 215: 1090–1101 www.newphytologist.com
pCAMBIA1300-35S-eGFP vector between the KpnI and Sal I sites, and fused in-frame to the N-terminus of enhanced green fluorescent protein (eGFP). The resulting constructs and pCAMBIA1300-35S-eGFP were transiently expressed in tobacco (Nicotiana benthamiana) leaves by Agrobacterium-mediated infiltration (strain EHA105), as described previously (Walter et al., 2004). The eGFP fluorescence of tobacco leaves was imaged 3 d after infiltration using a Zeiss LSM710NLO confocal laser scanning microscope. The excitation wavelength for eGFP fluorescence was 488 nm, and fluorescence was detected at 500– 542 nm. The primers are listed in Table S1. Analysis of total As and phosphorus (P) concentrations and As speciation Total As concentrations in plant samples were determined as described previously with minor modifications (Wu et al., 2011). Plant materials were digested with HNO3 : H2O2 (12 : 1, v/v). The total As concentration was determined using ICP-MS (DRC-e, Perkin-Elmer, Norwalk, CT, USA). Total P concentration was determined by inductively coupled plasma-optical emission spectrometry (ICP-OES; Optima 8000, Perkin-Elmer). Arsenic speciation in nutrient solutions, xylem saps and plant extracts was determined using HPLC-ICP-MS (NexIon 300x, Perkin-Elmer), as described previously (Shi et al., 2016).
Results Isolation and phenotypic characterization of the Oshac4 mutant To investigate the As(V) tolerance mechanism in rice, we screened an EMS-mutagenized library of rice with altered As(V) sensitivity using a root elongation assay. The mutant library was in the background of Osphf1-7, which is a weak mutant in OsPHF1 (Phosphate transporter facilitator 1) and is more tolerant than its WT (cv HJ2, Fig. S1) to As(V), because of decreased uptake of As(V) (Yang et al., 2016). The use of this mutant library is advantageous for the screening of putative As(V)hypersensitive mutants. One As(V)-hypersensitive mutant was obtained, showing significantly shorter root growth than the WT Osphf1-7 at both 10 lM and 20 lM As(V) (Fig. S1, without phosphate in the assay solution). We named this mutant Oshac4/ Osphf1-7 after the causal gene had been cloned. We also tested the sensitivity of Oshac4/Osphf1-7 to As(III) and found that Oshac4/Osphf1-7 had the same phenotype as Osphf1-7 and HJ2 (Fig. S2). The results suggested that the sensitivity was specific to As(V). After the causal gene for the mutant phenotype had been cloned, we backcrossed the Oshac4/Osphf1-7 mutant to HJ2 to segregate a single mutant Oshac4 in the HJ2 background. When grown in the normal nutrient solution (containing 100 lM phosphate) without As(V), there was no difference in the root elongation phenotype between HJ2 and Oshac4. After exposure to 10 or 20 lM As(V), the root length of 5-d-old Oshac4 was inhibited by 36.2% and 80.5%, respectively, compared with only 9% and Ó 2017 The Authors New Phytologist Ó 2017 New Phytologist Trust
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40.6% inhibition, respectively, in HJ2 (Fig. 1). The shoot length of Oshac4 was slightly more sensitive than that of HJ2 to As(V) in this short-term assay. Cloning and complementation test of Oshac4 We used genomic resequencing mapping to clone the mutant gene. The double mutant Oshac4/Osphf1-7 was backcrossed to the Osphf1-7 mutant, and seedlings of the F2 population were grown in a culture solution containing 20 lM As(V) for 5 d. Amongst the F2 population, 74 and 25 plants showed Osphf1-7 and mutant phenotypes, respectively, which is not significantly different from a 3 : 1 ratio (v2 = 0.02, P > 0.05). These results indicate that the phenotype of the mutant was caused by a recessive mutation. After genomic resequencing of 25 individual plants with mutant phenotypes, a point mutation (G975A) was found in the third intron of LOC_Os02g06290 (Fig. 2a,b), which was previously named OsHAC4 based on the sequence homology with AtHAC1, OsHAC1;1 and OsHAC1;2 (Shi et al., 2016). Because GT-AG is the splice site of the intron, the change in the nucleotide (G975A) causes an incorrect splice, resulting in the coding DNA sequence (CDS) of Oshac4 being 26 bp longer than that of OsHAC4 (Figs 2a,b, S3). To verify that the mutant phenotype was caused by the point mutation of OsHAC4, Oshac4 plants were transformed with
Flag-OsHAC4 under the control of its native promoter. Two independent transgenic lines were confirmed by PCR methods (Fig. 2c). At 20 lM As(V), root elongation of the two transgenic plants was rescued to the level of the WT HJ2 (Fig. 2d–f). These results indicate that the sensitivity to As(V) of the mutant Oshac4 is caused by the loss of function of OsHAC4. OsHAC4 has As(V) reductase activity OsHAC4 contains a rhodanase-like domain and shares a high similarity in its amino acid sequence with the As(V) reductases OsHAC1;1 and OsHAC1;2 (89% and 81%, respectively) (Shi et al., 2016). To test whether OsHAC4 can reduce As(V) to As (III), we expressed OsHAC4 in a strain of E. coli lacking the endogenous arsenate reductase ArsC (Oden et al., 1994). This mutant strain (DarsC) is hypersensitive to As(V) because it cannot reduce As(V) to As(III) to allow the latter to be extruded to the external medium. For comparison, OsHAC1;1 was also expressed in the DarsC mutant strain. With 1 mM As(V) in LB medium, the WT strain of E. coli grew normally, but the DarsC strain did not (Fig. 3a). The expression of either OsHAC4 or OsHAC1;1 restored the growth of the DarsC strain to that of WT (Fig. 3a). Furthermore, we analysed As speciation in the medium using HPLC-ICP-MS after E. coli strains had been exposed to 10 lM As(V) for 72 h. As(III) was produced by the DarsC strain
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Fig. 1 Phenotype of the Oshac4 mutant. (a) Five-day-old seedlings of wild-type (HJ2) rice (Oryza sativa) and the Oshac4 mutant grown in a nutrient solution containing 100 lM phosphate and different concentrations of arsenate. Bars, 2 cm. (b, c) Root (b) and shoot (c) length of HJ2 and Oshac4 plants exposed to different arsenate concentrations. Data represent means SD (n = 4). Significant difference from the corresponding WT: **, P < 0.01. Ó 2017 The Authors New Phytologist Ó 2017 New Phytologist Trust
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expressing either OsHAC4 or OsHAC1;1, but not by the DarsC strain with the empty vector (Fig. 3b). The magnitude of As(III) production was comparable between OsHAC4 and OsHAC1;1. These results confirm the ability of OsHAC4 to reduce As(V) to As(III). New Phytologist (2017) 215: 1090–1101 www.newphytologist.com
Fig. 2 Cloning of OsHAC4 and complementation analysis. (a) Mutation site and the sequence surrounding the point mutant of Oshac4. (b) DNA sequencing results of OsHAC4 in wild-type (HJ2) rice (Oryza sativa) and the Oshac4 mutant. The red arrow indicates the mutation position. (c) PCR analysis using the coding DNA sequence (CDS) primer (top) and a cleaved amplified polymorphic sequence (CAPS) marker (bottom) for HJ2, the Oshac4 mutant and two transgenic plants transformed with POsHAC4::Flag:OsHAC4. (d) Phenotypes of 5-d-old plants of HJ2, Oshac4 and two transgenic plants transformed with Flag: OsHAC4 after growth in nutrient solution without (d) or with (e) 20 lM arsenate. Bars, 2 cm. (f) Root length of plants in (d) and (e), respectively. Data represent means SD (n = 4). Different letters above the bars represent significant differences at P < 0.05.
Temporal and spatial expression patterns of OsHAC4 There are > 10 AtHAC1-like genes in the rice genome (Shi et al., 2016). We selected five other OsHAC genes with a high homology to OsHAC4 for expression analysis (Fig. S4). The total RNA Ó 2017 The Authors New Phytologist Ó 2017 New Phytologist Trust
New Phytologist of different tissues of HJ2 was extracted to investigate the expression pattern of OsHAC genes. Quantitative RT-PCR analysis showed that OsHAC4 was predominantly expressed in roots, with little expression in other tissues (Fig. 4a). Among the six OsHAC genes quantified, OsHAC4 showed the highest transcript abundance in roots. Previously, Shi et al. (2016) have shown that OsHAC1;1 is expressed in both roots and shoots. Our results showed that OsHAC1;1 was also highly expressed in stems and nodes. In addition, OsHAC2 and OsHAC7 also showed high levels of expression in stems and nodes (Fig. 4a). To determine the tissue expression pattern of OsHAC4, we generated rice (cv HJ2) transgenic lines with the GUS reporter gene driven by a 3.4-kb fragment native promoter of OsHAC4. Histochemical analysis of OsHAC4pro-GUS transgenic plants
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revealed that OsHAC4 expression was visible in roots (Fig. 4b), whereas no signals were observed in leaves (Fig. S5). Crosssections of the primary root showed that OsHAC4 was expressed exclusively in the epidermal and exodermal cells of the root tip, the elongation zone and the maturation zone (Fig. 4b). To determine whether OsHAC4 expression was regulated by As (V) exposure, the transcript level of OsHAC4 in roots was analysed by quantitative RT-PCR. Twenty-day-old WT (HJ2) seedlings were exposed to different concentrations (0–50 lM) of As(V) for 12 h, or to 10 lM As(V) for 0–24 h. Phosphate was withdrawn from the nutrient solution during As(V) exposure to facilitate As (V) uptake by roots. The level of OsHAC4 transcript in roots was up-regulated by different As(V) concentrations, with a five-fold increase in the 50 lM As(V) treatment (Fig. 4c). Time course analysis showed that exposure to 10 lM As(V) significantly enhanced the expression of OsHAC4 during the first 12 h (Fig. 4d). Subcellular localization of OsHAC4 To investigate the subcellular localization of OsHAC4, we constructed an OsHAC4-eGFP fusion protein driven by the cauliflower mosaic virus 35S promoter and expressed it transiently in tobacco (Nicotiana benthamiana) leaf cells. The GFP signal of OsHAC4-eGFP fusion protein was observed in the cytoplasm and nucleus (Fig. 5). This subcellular localization is similar to that of OsHAC1;1 and OsHAC1;2 (Shi et al., 2016). OsHAC4 mutation leads to decreased As(V) reduction and As(III) efflux, and increased As accumulation in plant roots and shoots
Fig. 3 OsHAC4 encodes an arsenate reductase. (a) Expression of OsHAC4 or OsHAC1;1 suppresses the As(V) sensitivity of the Escherichia coli mutant lacking the arsC arsenate reductase. WT, wild-type. (b) Production of As(III) in Luria–Bertani (LB) medium by E. coli mutant expressing the empty vector (DarsC), OsHAC1;1 or OsHAC4. Data represent means SE (n = 4). Ó 2017 The Authors New Phytologist Ó 2017 New Phytologist Trust
To investigate whether OsHAC4 is involved in As(V) reduction and As accumulation in rice, Oshac4 mutant and WT plants were exposed to 10 lM As(V) in the absence of phosphate for 48 h. Arsenic speciation analysis using HPLC-ICP-MS showed that Oshac4 roots accumulated more As(V) and As(III) than WT roots, with 2.3- and 1.3-fold differences for the two As species, respectively (Fig. 6a). The percentage of As(III) in the total As in roots, reflecting the As(V) reduction activity in roots, decreased significantly from 82% in WT to 72% in the mutant. In shoots, Oshac4 accumulated a three-fold higher As(III) concentration than WT, whereas there was no difference in the As(V) concentration between Oshac4 and WT (Fig. 6b). The shoot to root As concentration ratio increased from 0.03 in WT to 0.05 in the mutant. In a further experiment, plants were exposed to 5 lM As(V) in the presence of 100 lM phosphate for 2 h and 24 h, and total As concentrations in roots and shoots were determined. Compared with WT, Oshac4 showed 5.4- and 1.4-fold higher total As concentration in shoots and roots, respectively, after 2 h of As(V) exposure (Fig. S6). After 24 h, the difference between Oshac4 and WT in terms of the total As concentration in shoots increased to nine times. At both time points, the shoot to root As concentration ratio of Oshac4 (0.055–0.126) was markedly higher than that of WT (0.014–0.016). There was no significant difference between the mutant and WT in either root or shoot P concentration (Fig. S7). New Phytologist (2017) 215: 1090–1101 www.newphytologist.com
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Fig. 4 Expression pattern of OsHAC4 in wildtype (HJ2) rice (Oryza sativa). (a) Quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) analysis of OsHACs in different rice tissues. Expression of each gene was calculated as 2DCT relative to OsActin. (b) bGlucuronidase (GUS) staining of OsHAC4pro-GUS transgenic plants observed in: (i) primary root; the cross-section of (ii) root elongation zone where the lateral roots emerge, (iii) root mature zone and (iv) root tip; (v) lateral root. Bars: (i, v) 0.5 mm; (ii, iii, iv) 50 lm. (c, d) qRT-PCR analysis of OsHAC4 in the root of wild-type plants grown in solution with different concentrations of As(V) for 12 h (c) and with 10 lM As(V) for different times (d). Data represent means SE (n = 4).
Fig. 5 Subcellular localization of OsHAC4. Representative microscopic images of tobacco (Nicotiana benthamiana) epidermal cells expressing the OsHAC4:eGFP fusion protein or enhanced green fluorescent protein (eGFP) driven by the cauliflower mosaic virus 35S promoter.
Previous studies by Chao et al. (2014) and Shi et al. (2016) have shown that AtHAC1, OsHAC1;1 and OsHAC1;2 function as As(V) reductases in the roots of A. thaliana and rice, New Phytologist (2017) 215: 1090–1101 www.newphytologist.com
respectively, enabling the product of As(V) reduction, As(III), to be extruded to the external medium, and thus limiting As accumulation in both roots and shoots. To test whether mutation in Ó 2017 The Authors New Phytologist Ó 2017 New Phytologist Trust
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sap was analysed. The concentrations of both As(V) and As(III) in the xylem sap from the Oshac4 mutant were significantly higher, by 2.2- and 6.2-fold, respectively, than those from WT (Fig. 6e). The percentage of As(III) in the xylem sap total As was lower in the WT (19%) than in the Oshac4 mutant (40%). Overexpression of OsHAC4 enhances As(V) tolerance and decreases As accumulation To further investigate the role of OsHAC4 in As(V) tolerance and As accumulation, we overexpressed OsHAC4 in rice (cv HJ2) using the cauliflower mosaic virus 35S promoter. Two independent lines with the overexpression of OsHAC4 were selected for further characterization. Quantitative RT-PCR analysis showed that both lines exhibited greatly enhanced expression of OsHAC4 in leaves compared with HJ2 plants (Fig. 7a). After exposure to 20 lM As(V) for 5 d, the root lengths of the two OsHAC4OVER lines were significantly longer than those of the HJ2 plants, whereas the Oshac4 mutant showed only approximately one-half of the root length of WT (Fig. 7b,c). To investigate the function of OsHAC4 in As accumulation, total As concentrations in roots and shoots of OsHAC4-OVER and WT lines were determined after plants had been exposed to 10 lM As(V) in a nutrient solution without phosphate for 6 and 48 h. Both OsHAC4-OVER lines had significantly lower As concentrations than WT plants in both shoots and roots, with a decrease of 39.2–42.1% in root As concentration and of 38.2– 40% in shoot As concentration, respectively (Fig. 7d,e).
Fig. 6 Altered arsenate reduction and arsenite efflux in Oshac4. (a, b) As speciation in roots (a) and shoots (b) after wild-type (HJ2) rice (Oryza sativa) and Oshac4 mutant plants had been exposed to 10 lM As(V) for 48 h. (c, d) Uptake of As(V) (c) and efflux of As(III) (d) after HJ2 and Oshac4 mutant plants had been exposed to 10 lM As(V) for 48 h. FW, fresh weight. (e) Concentrations of As(V) and As(III) in xylem sap of HJ2 and Oshac4 mutant plants. Plants were exposed to 10 lM As(V) for 24 h. Data are means SE (n = 4). Significant difference from the corresponding WT: **, P < 0.01.
OsHAC4 also affects As(III) efflux in rice, plants were exposed to 10 lM As(V) for 48 h. As(V) uptake and As(III) efflux were estimated by measuring the changes in As speciation in the culture solution. There was no significant difference in As(V) uptake between the mutant and WT (Fig. 6c). By contrast, As(III) efflux from roots to the external medium decreased significantly in Oshac4 compared with WT (Fig. 6d). WT plants extruded 74% of the As taken up as As(V) in the form of As(III) to the external medium during the 48-h period. By contrast, Oshac4 released only 54% of the As(V) taken up by roots. These results show that mutation in OsHAC4 affects As(V) reduction in roots and the subsequent As(III) efflux to the external medium. OsHAC4 mutation leads to increased As concentration and altered As speciation in xylem sap Xylem sap was collected from WT and Oshac4 plants after exposure to 10 lM As(V) for 24 h. Arsenic speciation in the xylem Ó 2017 The Authors New Phytologist Ó 2017 New Phytologist Trust
Discussion As(V) reduction is the first step of As(V) detoxification in plants. Recent studies have identified AtHAC1 (AtATQ1) as a new type of As(V) reductase that plays an important role in As(V) tolerance and in the control of As accumulation in A. thaliana (Chao et al., 2014; Sanchez-Bermejo et al., 2014). Using a reverse genetics approach, Shi et al. (2016) showed that two rice orthologues of AtHAC1, OsHAC1;1 and OsHAC1;2, also function as As(V) reductases and regulate As accumulation in rice. In the present study, we used a forward genetics approach with the aim of identifying the components of As(V) tolerance in rice. We isolated an As(V)-sensitive mutant that exhibited a much greater inhibition than WT of root elongation by As(V). This phenotype was consistent regardless of whether the mutation was in the Osphf1-7 or HJ2 background (Figs 1, S1). By contrast, the mutant was no more sensitive to As(III) than WT, indicating that the mutation does not affect the detoxification of As(III). Using genomic resequencing and a complementation test, we identified OsHAC4 as the casual gene for the mutant phenotype (Fig. 2). Similar to AtHAC1, OsHAC1;1 and OsHAC1;2, OsHAC4 encodes a rhodanase-like protein, but its function has not been investigated previously. Using a heterologous expression system, we showed that OsHAC4 is able to confer As(V) tolerance in the E. coli DarsC strain lacking the endogenous As(V) reductase and can reduce As(V) to As(III) (Fig. 3), confirming OsHAC4 as an As(V) reductase. This function is similar to AtHAC1, New Phytologist (2017) 215: 1090–1101 www.newphytologist.com
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Fig. 7 Overexpression of OsHAC4 enhances arsenate tolerance. (a) The expression levels of OsHAC4 in leaves of wild-type (HJ2) rice (Oryza sativa) and transgenic lines by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). (b) Five-day-old seedlings of HJ2, Oshac4 mutants and OsHAC4-OVER lines grown in solution with 20 lM As(V). (c) Root length of plants described in (b). (d, e) Total As concentrations in roots and shoots after HJ2 and OsHAC4-OVER lines had been exposed to 10 lM As(V) for 6 h (d) or 48 h (e). DW, dry weight. Data represent means SE (n = 4). Different letters above bars represent significant difference at P < 0.05.
OsHAC1;1 and OsHAC1;2 (Chao et al., 2014; Shi et al., 2016). In planta, OsHAC4 mutation leads to an accumulation of As(V) and a decrease in the proportion of As(III) in the total As concentration in roots, indicating that OsHAC4 plays a role in As(V) reduction in rice roots (Fig. 6). Although As(V) uptake was unaffected, As(III) efflux to the external medium was significantly lower in the Oshac4 mutant (Fig. 6). The decreased As(III) efflux following As(V) uptake is consistent with the loss of function of OsHAC4 as an As(V) reductase. The decreased As(III) efflux also explains the phenotype of increased As accumulation in both roots and shoots of the mutant (Fig. 6), which would, in turn, result in increased As(V) sensitivity. As(III) efflux typically accounts for 60–80% of the As(V) taken up by plant roots (Xu et al., 2007; Liu et al., 2010; Zhao et al., 2010a). As(V) reductases are an essential component of this detoxification mechanism because the enzymes transform the phosphate analogue As(V) to As(III), an As species that is chemically dissimilar to phosphate. As(III) can then be extruded out of the cell via transporters, such as aquaporin channels (Zhao et al., 2010a), thus decreasing the cellular burden of As without the risk of losing cellular phosphate. New Phytologist (2017) 215: 1090–1101 www.newphytologist.com
Although the function of OsHAC4 appears to be similar to that of OsHAC1;1 and OsHAC1;2, there are differences in their expression patterns in rice tissues. OsHAC4 is predominantly expressed in roots, with the transcript abundance being the highest amongst the six OsHAC genes quantified, including OsHAC1;1 and OsHAC1;2 (Fig. 4). At the cellular level, OsHAC4 is strongly and exclusively expressed in the epidermis and exodermis of roots (Fig. 4), a location that is ideally suited for the efflux of As(III) to the external medium following As(V) uptake. The fact that OsHAC4 expression is induced by As(V) exposure (Fig. 4) is also consistent with its role in As(V) detoxification. In comparison, OsHAC1;1 is expressed in the epidermis and the pericycle cells, whereas OsHAC1;2 is expressed in the outer layers of the cortex and the endodermis cells of roots (Shi et al., 2016). In addition, OsHAC1;1 is also strongly expressed in stems and nodes (Fig. 4). The differences in the expression levels and in the cellular patterns of expression probably explain why the Oshac4 mutant shows a strong phenotype of As(V) sensitivity (Fig. 1), whereas Oshac1;1 and Oshac1;2 single mutants do not (Shi et al., 2016). These results indicate that OsHAC4 plays a much more important role in As(V) detoxification than does Ó 2017 The Authors New Phytologist Ó 2017 New Phytologist Trust
New Phytologist OsHAC1;1 or OsHAC1;2 individually. The present study and that of Shi et al. (2016) showed that ectopic overexpression of OsHAC4, OsHAC1;1 or OsHAC1;2 enhances As(V) tolerance in rice (Fig. 7). It is possible that this effect results primarily from the overexpression of the As(V) reductases in the outer layer of the root cells driven by a constitutive promoter. Based on the present study and that of Shi et al. (2016), there are at least three As(V) reductases in rice. Several other OsHAC homologues may also serve as As(V) reductases, although their functions remain to be investigated. The different As(V) reductase isoforms may have some degree of functional redundancy, as Shi et al. (2016) showed that the double mutant Oshac1;1 Oshac1;2 exhibited a stronger phenotype than the single mutants. However, the strong phenotype of the Oshac4 mutant suggests that any functional redundancy of OsHAC4 with other isoforms is likely to be small, at least in terms of As(V) detoxification. Because of the presence of multiple As(V) reductases and possibly also non-enzymatic mechanisms of As(V) reduction in rice, it is not surprising that As(III) is still the main As species in the roots and shoots of the Oshac4 mutant exposed to As(V) (Fig. 6). Indeed, the mutant roots contain higher amounts of not only As (V), but also As(III). In mutant shoots, increased As accumulation is primarily in the form of As(III). It is likely that different As(V) reductases catalyse As(V) reduction in different tissues or cell types depending on their localization, with OsHAC4 contributing mainly to As(V) reduction in the epidermal and exodermal cells of roots. It is intriguing that the rice genome contains > 10 OsHAC homologue genes (Shi et al., 2016), whereas A. thaliana has only one gene (Chao et al., 2014). This difference explains why AtHAC1 mutation produces a far greater effect on As accumulation than single mutations of OsHAC4, OsHAC1;1 or OsHAC1;2. Although the function of AtHAC1 and OsHACs as As(V) reductases is clear, it is not known whether they also possess other functions. Excessive As accumulation in rice grain poses a health risk to rice consumers (Meharg et al., 2009; Zhao et al., 2010b). It is therefore important to find solutions to minimize As accumulation in rice. The present study shows that overexpression of OsHAC4 not only enhances As(V) tolerance, but also decreases significantly As accumulation in rice shoots (Fig. 7). Shi et al. (2016) reported similar results with overexpression of OsHAC1;1 or OsHAC1;2. It should be noted that these results were obtained in experiments in which rice plants were exposed to As(V). As (III) is the predominant form of As in flooded paddy soils in which reducing conditions prevail, although As(V) still accounts for 10–30% of the soluble As in soil solution (Khan et al., 2010). The proportion of As(V) in the rhizosphere may be even higher as a result of oxygen release from the aerenchyma of rice roots (Seyfferth et al., 2010). Paddy water is drained episodically during the rice growing season, resulting in aerobic conditions in the paddy soil, which promote the oxidation of As(III) to As(V). There is also an increasing trend of growing aerobic rice to save water (Bouman et al., 2005). Enhancing the activity of OsHAC4 could provide a useful option for the limitation of As accumulation in rice crops that experience long periods of aerobic conditions. It would also be interesting to investigate whether there is Ó 2017 The Authors New Phytologist Ó 2017 New Phytologist Trust
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allelic variation in OsHAC4 that can explain varietal differences in As(V) tolerance or As accumulation. In conclusion, we have identified OsHAC4 as a new As(V) reductase that is critical for As(V) tolerance and As accumulation in rice. Its role in As(V) tolerance and As accumulation is underpinned by the high expression level of the gene, as well as the localization of expression exclusively in the epidermis and exodermis of rice roots, where the product of As(V) reduction can be easily extruded to the external medium to decrease the cellular burden of As. Our study provides new insights into the mechanisms of As(V) detoxification and As accumulation in rice.
Acknowledgements We thank Mingxiu Chen for developing the transgenic plants, and Zhu Tang for arsenic speciation analysis. This work was supported by the National Key Research and Development Program of China (2016YFD0100700), National Natural Science Foundation of China (31520103914 to F-J.Z. and 31570244 to Z.W.), and the Natural Science Foundation of Zhejiang Province (LY16C020001).
Author contributions Z.W. and F-J.Z. designed the research. J.X., S.S., L.W., Z.T., T.L., X.Z., X.D. and Y.W performed the experiments. J.X., Z.W. and F-J.Z. analysed the data. F-J.Z., Z.W. and J.X. wrote the paper with contributions from all the authors.
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Supporting Information Additional Supporting Information may be found online in the Supporting Information tab for this article:
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Fig. S3 The coding DNA sequence (CDS) of Oshac4 is 26 bp longer than that of OsHAC4 in wild-type (HJ2) rice (Oryza sativa). Fig. S4 Phylogenetic analysis of seven HAC genes in rice (Oryza sativa) and Arabidopsis thaliana. Fig. S5 b-Glucuronidase (GUS) staining of OsHAC4 in leaves of transgenic rice (Oryza sativa). Fig. S6 OsHAC4 mutation affects arsenic accumulation in rice (Oryza sativa). Fig. S7 OsHAC4 mutation has no significant effect on phosphorus concentration in rice (Oryza sativa). Table S1 The primers used in this study
Fig. S1 Phenotypic characterization of the rice Oshac4/Osphf1-7 mutant in response to As(V) exposure. Fig. S2 Phenotypic characterization of the rice Oshac4/Osphf1-7 mutant in response to As(III) exposure.
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