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. 2021 Feb 22;186(1):611–623. doi: 10.1093/plphys/kiab086

Deficiency in alcohol dehydrogenase 2 reduces arsenic in rice grains by suppressing silicate transporters

Shimpei Hayashi 1, Masato Kuramata 2, Tadashi Abe 2, Noriko Yamaguchi 2, Hiroki Takagi 3, Hachidai Tanikawa 2, Manaka Iino 2, Kazuhiko Sugimoto 4, Satoru Ishikawa 2,✉,2
PMCID: PMC8154085  PMID: 33620496

Abstract

Paddy fields are anaerobic and facilitate arsenite (As(III)) elution from the soil. Paddy-field rice accumulates arsenic (As) in its grains because silicate transporters actively assimilate As(III) during the reproductive stage. Reducing the As level in rice grains is an important challenge for agriculture. Using a forward genetic approach, we isolated a rice (Oryza sativa) mutant, low arsenic line 3 (las3), whose As levels were decreased in aerial tissues, including grains. The low-As phenotype was not observed in young plants before heading (emergence of the panicle). Genetic analyses revealed that a deficiency in alcohol dehydrogenase (ADH) 2 by mutation is responsible for the phenotype. Among the three rice ADH paralogues, ADH2 was the most efficiently produced in root tissue under anaerobic conditions. In wild-type (WT), silicon and As concentrations in aerial tissues increased with growth. However, the increase was suppressed in las3 during the reproductive stage. Accordingly, the gene expression of two silicate transporters, Lsi1 and Lsi2, was increased in WT around the time of heading, whereas the increase was suppressed in las3. These results indicate that the low-As phenotype in las3 is due to silicate transporter suppression. Measurement of intracellular pH by 31P-nuclear magnetic resonance revealed intracellular acidification of las3 roots under hypoxia, suggesting that silicate transporter suppression in las3 might arise from an intracellular pH decrease, which is known to be facilitated by a deficiency in ADH activity under anaerobic conditions. This study provides valuable insight into reducing As levels in rice grains.

Deficiency in alcohol dehydrogenase suppresses arsenite uptake via silicate transporters and reduces arsenic levels in rice grains.

Introduction

Arsenic (As) is an environmental pollutant existing in nature. Inorganic As compounds tend to adhere to soils as arsenate (As(V)) under aerobic (oxidative) conditions (Takahashi et al., 2004). Under anaerobic reductive conditions, As(V) is reduced and eluted to soil water as arsenite (As(III)). Paddy fields are anaerobic and thereby facilitate the elution of As(III) from the soil. Paddy rice (Oryza sativa) actively assimilates As(III) through silicate transporters during the reproductive stage and accumulates As in grains (Ma et al., 1989, 2006, 2007, 2008). Due to these factors, rice grains are a major dietary source of As in humans (Schoof et al., 1999; Oguri et al., 2014). Since As poses a serious health risk to living organisms (World Health Organization, 2011), reducing As levels in rice grains is an important challenge for agriculture.

Several approaches for reducing As in rice grains have been proposed and reviewed (Ishikawa et al., 2019; Kumarathilaka et al., 2020; Zhao and Wang, 2020). Many studies have demonstrated that alternate wetting and drying water practices are effective for reducing As dissolution from the paddy soil and As accumulation in rice grains (Arao et al., 2009; Ishikawa et al., 2016; Honma et al., 2016a). Some fertilizers and amendments are also useful for reducing As uptake in rice. For example, application of iron (Fe)-bearing materials to paddy soil can directly immobilize As and reduce As(III) release into the soil solution, resulting in reduced As uptake by rice roots (Makino et al., 2016; Honma et al., 2016b). Knowledge on the genetic and molecular aspects of As reduction in rice has gradually accumulated to facilitate the breeding of low-As cultivars.

Rice roots are capable of taking up large amounts of silicate and As(III) from paddy soil solutions. In roots, the transfer of water-soluble compounds to vascular tissues is blocked by the Casparian strip, which is a hydrophobic barrier around the exodermis and endodermis (Enstone et al., 2002). Silicate passes through barriers via the transmembrane pathway mediated by the plasma membrane-localized silicate transporters OsLsi1 and OsLsi2 (Ma and Yamaji, 2008). OsLsi1 and OsLsi2 are polarly localized on the distal and proximal sides of both exodermal and endodermal cells, respectively (Ma et al., 2011). OsLsi1 is an aquaporin-like influx transporter, whereas OsLsi2 is an efflux transporter driven by the proton gradient (Ma and Yamaji, 2008). OsLsi1 and OsLsi2 can also mediate As(III) influx in root cells and As(III) efflux toward xylem cells, respectively (Ma et al., 2008). Gene expression of OsLsi1 and OsLsi2 is regulated by plant development and some physiological and biochemical factors (Yamaji and Ma, 2007, 2011). For instance, their expression is induced by depletion of silicate and suppressed by excess silicate (Yamaji and Ma, 2007, 2011). Additionally, the phytohormone abscisic acid suppresses their expression (Yamaji and Ma, 2007, 2011). Since silicate is important for rice development, null mutations of OsLsi1 and OsLsi2 have significantly negative effects on the growth and defense of plants (Ma and Yamaji, 2006), but positive effects on decreasing As(III) for root uptake and accumulation in grains (Ma et al., 2008).

Paddy rice can adapt to waterlogged anaerobic conditions by the development of root aerenchyma and shift to fermentative metabolism. Fe-plaque on the root surface is formed by radial oxygen release through the root aerenchyma and can sequester As (Wu et al., 2012), although the contribution of Fe-plaque to inhibiting As uptake is still under debate. In vascular plants, alcoholic fermentation is necessary for germination and survival under anaerobic conditions (Saika et al. 2006), and alcohol dehydrogenase (ADH) is one of key enzymes for anaerobic tolerance in rice (Matsumura et al. 1995). However, involvement of ADH in As(III) uptake of anaerobic rice has not been reported.

In this study, we isolated a rice mutant ( low arsenic line 3 [las3]) with a mutation in OsADH2, which has a significantly lower As concentration in its grains than the wild-type (WT). We report the characterization of las3 and discuss the mechanism responsible for the low-As phenotype.

Results

Isolation of the las3 mutant

A mutant, named las3, with a low level of As in its grains, was screened from M2 progeny of a mutagenized japonica cultivar “Koshihikari.” The phenotype of las3 was also evaluated in progeny lines grown under different water regimes in a paddy field. Flooded irrigation increased the As concentrations in grains and straws of both WT and las3 plants, whereas water-saving irrigation substantially decreased those concentrations (Figure 1A). As concentrations in the grains of las3 decreased by ∼30% compared to those of WT (Figure 1A) in all water regimes. Additionally, the As concentration in the straw of las3 decreased by >60% compared to that of the WT (Figure 1A). In straws of las3, As concentrations were decreased in all of the organs (Figure 1B). We could not measure accurate As levels in roots at the same time because it was difficult to remove soil particles from roots. However, these data indicate that the functions of roots in las3 are distinct from those of WT. We also measured As levels in aerial and root tissues of young plants grown in hydroponic culture. However, the As levels were similar between WT and las3 (Supplemental Figure S1). These data indicate that the low-As phenotype of las3 is expressed during a particular period of growth. The culm length, grain weight, and straw weight of las3 were slightly lower than those of WT (Figure 1C;  Supplemental Table S1).

Figure 1.

Figure 1

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Phenotypes of the las3 mutant. (A) As contents in grains and straw harvested from WT and las3 mutant plants grown under three water regimes in a paddy field containing 1.4 mg kg−1 As. (B) As contents in individual parts of aerial tissues in plants grown under flooded irrigation. (C) Morphology of WT and las3 plants grown under flooded irrigation. Data are the means ± SDs of three replicates. **P < 0.01, *P < 0.05 (t test).

Identification of the las3 mutation

F2 progeny (94 plants) obtained by crossing las3 with the indica rice cultivar “Habataki” were genotyped by using molecular markers consisting of single-nucleotide polymorphisms (SNPs) and measured As levels in grains, husks, and straws sampled from the F2 plants cultivated in anaerobic paddy soil. The logarithm of odds score in the quantitative trait locus analysis indicated that the low-As phenotypes observed in common for grains, husks, and straws were tightly linked to a single region on chromosome 11 (Supplemental Figure S2A). Graphical genotype with SNP markers indicated that a candidate gene was located within a 2.7-Mbp region on chromosome 11 (Supplemental Figure S2B). Whole-genome sequencing of the las3 mutant revealed that the mapped region contained a single mutation, namely a single-base substitution in the OsADH2 gene (Os11g0210500). This mutation replaced cysteine 99 with a phenylalanine residue in OsADH2 (Figure 2A). The mutated cysteine residue is widely conserved in the ADH family and is known as a component of a zinc-binding site that contributes to stabilization of the protein structure (Strommer, 2011; Magonet et al., 1992; Figure 2B;  Supplemental Figure S2C). Therefore, the mutated OsADH2 in las3 is likely to be unstable and dysfunctional due to the disruption of the zinc-binding site. The metal-binding sites of proteins are important interaction sites for As (Hughes, 2002). To investigate if the mutation affects the interaction between the zinc-binding site and As, we evaluated allelic mutants of las3. One of the allelic mutants is a null mutant in which the Tos17 transposon was inserted into the fourth exon of OsADH2. The other is a missense mutant in which glycine 207 was replaced with an aspartic acid residue and whose ability to bind nicotinamide adenine dinucleotide, which is required for enzymatic activity, was lost. This mutant was selected from a mutant library of Japonica rice cultivar “Koshihikari” using a Targeting Induced Local Lesions IN Genomes (TILLING) method. Measurements of As in grains and straws demonstrated that these allelic mutants showed a low-As phenotype similar to that of the las3 mutant (Figure 2, C and D). This result indicates that the cause of the low-As phenotype is a deficiency in enzymatic activity but not changes in interactions with As. The similar phenotype among the alleles genetically demonstrated that OsADH2 is the gene responsible for the las3 mutant phenotype.

Figure 2.

Figure 2

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Identification of the las3 mutation. (A) Schematic representation of the las3 locus. Black and white boxes indicate exons and untranslated regions, respectively. A single-base substitution occurred in the Os11g0210500 (OsADH2) gene. The mutation replaced cysteine 99 with a phenylalanine residue. (B) Alignment of amino acid sequences around the structural zing binding sites in Arabidopsis thaliana ADH1 and OsADH2. (C) As contents in grain and straw harvested from a Tos17-insertion disruptant of OsADH2 (Tos17). “Koshihikari” and “Nipponbare” were used as WT of las3 and Tos17, respectively. The plants were grown in pots filled with soil with a relatively high As level. Data are the means ± SDs of three replicates. **P < 0.01 (t test). (D) As contents in grains and straw harvested from the G207D allele mutant, in which glycine 207 is replaced with an aspartic acid, causing it to lose its enzymatic activity. The plants were grown in a paddy field under submerged conditions. Data are the means ± SDs of four replicates. Bars with different letters differ significantly (Tukey’s test, P < 0.05).

Comparison among rice ADH paralogues

O. sativa has at least three ADH paralogue genes (OsADH1, Os11g0210300; OsADH2, Os11g0210500; and OsADH3, Os11g0210600) that are closely located on chromosome 11. These translation products are thought to have similar molecular functions because their amino acid sequences are ∼80% identical (Supplemental Figure S3). Although OsADH1 is known to be involved in coleoptile elongation under submergence (Saika et al., 2006), the roles of OsADH2 and OsADH3 have not been reported. To investigate whether mutations of OsADH1 and OsADH3 show a low-As phenotype similar to that of OsADH2, we generated OsADH1 and OsADH3 disruptants by genome editing (Supplemental Figure S4A) and measured the As concentration in the grains. These disruptants did not show a low-As phenotype (Figure 3A). A similar result was also observed in a well-known allelic mutant of OsADH1 (rad; Supplemental Figure S4B; Saika et al., 2006). These results indicate that some individual properties of OsADH2 are involved in the expression of the low-As phenotype of las3. To evaluate redundancy among the ADH paralogues in rice roots, we first compared the copy numbers of their transcripts. Since the expression of ADHs is known to be induced by anaerobic treatments (Xie and Wu, 1989), rice seedlings were incubated in the AnaeroPack system that works as an oxygen absorber-carbon dioxide generator to provide and maintain anaerobic conditions in an airtight box (Delaney and Onderdonk, 1997). In WT roots, with or without anaerobic treatments, the copy numbers of OsADH1 and OsADH2 transcripts were predominantly high compared to those of OsADH3 transcripts (Figure 3B). The expression of all of the rice ADHs was induced by anaerobic treatment, although the expression level of OsADH3 was very low. When the expression levels of three ADHs were compared between WT and las3 roots under hypoxia, a significant difference was not observed at each treatment time (Figure 3C). Next, we investigated the amounts of translation products of the rice ADHs in roots. To detect endogenous ADHs, we obtained an antibody that recognized OsADH1, OsADH2, and OsADH3 almost equally (Supplemental Figure S5) and total proteins of ADHs were analyzed by immunoblot. In the WT, the accumulation level of total ADHs was increased by anaerobic treatment. However, the accumulation in las3 was markedly lower than that in WT (Figure 3D), although transcript levels were similar between WT and las3 (Figure 3C). This result indicates that in rice ADH paralogues, OsADH2 was most efficiently produced in root tissue under anaerobic conditions. Additionally, these results suggest that las3 mutation inhibits the accumulation of translation products, probably due to a decrease in protein stability.

Figure 3.

Figure 3

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Comparison among rice ADH paralogues. (A) As contents in grains harvested from las3 and OsADH1 (adh1) and OsADH3 (adh3) disruptants. The plants were grown under continuously submerged conditions in pots filled with soil with a relatively high As level. Data are the means ± SDs of three replicates. Bars with different letters differ significantly (Tukey’s test, P < 0.05). (B) mRNA levels of OsADH1 and OsADH2 (left panel), and OsADH3 (right panel) in roots of 7-d-old WT seedlings after anaerobic treatment. Copy numbers of the cDNAs synthesized from 1 ng of total RNA are shown. RT-qPCR analyses of three biological replicates. Means ± SDs. Bars with different letters differ significantly (Tukey’s test, P < 0.05). (C) Relative mRNA levels of OsADH1, OsADH2, and OsADH3 in roots of 7-d-old seedlings of WT and las3 after anaerobic treatment. The 25S rRNA gene was used as an internal reference. Data are the means ± SDs of three replicates. Bars with different letters differ significantly (Tukey’s test, P < 0.05). (D) Immunoblot analysis of rice ADHs in roots of 7-d-old seedlings after anaerobic treatment. The anti-ADH antibody detected OsADH1, OsADH2, and OsADH3 almost equally (Supplemental Figure S4). A blotting membrane stained with Coomassie Brilliant Blue is shown as a loading control. Similar results were obtained in at least three independent experiments.

Uptake of silicate and As(III) during the heading stage

In rice, uptake of As is facilitated, especially during the reproductive stage, and silicate transport largely contributes to As(III) uptake (Akahane et al. 2020). To investigate the involvement of silicate transport in the low-As phenotype of las3, we measured silicon (Si) and As levels in aerial tissues before and after the heading period (Figure 4A). Before heading (30 d after transplant), Si and As levels were similar between WT and las3. However, Si and As levels in las3 were significantly lower than those of WT at heading and mature (after heading) stages. The Si and As levels in the WT increased until maturity, whereas these increases were suppressed in las3. This result strongly suggests that the low-As phenotype in las3 is due to the suppression of silicate transport. In addition to silicate transport, phosphate (Pi) transport is known to contribute to As uptake, because As(V) is structurally similar to Pi (Catarecha et al., 2007). However, phosphorus (P) levels were similar between WT and las3 during plant growth (Supplemental Figure S6), indicating that reduced uptake of As(III) is the main cause of the low-As phenotype of las3.

Figure 4.

Figure 4

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Silicate and As uptake after transplanting. (A) Si and As contents in aerial tissues of WT and las3 plants after transplanting. One-month-old seedlings were transplanted to the paddy field, and 5 weeks (before heading), 11 weeks (at heading), and 16 weeks (after heading) after transplanting, plants were harvested. Data are the means ± SDs of three replicates. **P < 0.01 (t test). (B) mRNA levels of silicate transporter genes OsLsi1 and OsLsi2 in roots of WT and las3 plants. The roots were collected from the plants grown in the paddy field at 5 weeks (before heading), 11 weeks (at heading), and 14 weeks (after heading) after transplanting. Data are the means ± SDs of three replicates. **P < 0.01, *P < 0.05 (t test).

Expression levels of silicate transporters

The reduced transport of silicate suggests that the actions of silicate transporters were suppressed in las3. To investigate this possibility, we compared the gene expression levels of silicate transporters OsLsi1 (Os02g0745100) and OsLsi2 (Os03g0107300) between WT and las3 (Figure 4B). Consistent with previous reports (Yamaji and Ma, 2007, 2011), the transcript levels of OsLsi1 and OsLsi2 in WT increased at heading time. However, the increase was suppressed in las3 (Figure 4B). This expression pattern was consistent with Si and As level changes, suggesting that the suppression of OsLsi1 and OsLsi2 expression is a major cause of the lower Si and As levels in las3.

Expression change of ADHs

According to the Rice Expression Profile Database (RiceXPro; http://ricexpro.dna.affrc.go.jp), OsADH2 was expressed in all tissues of roots, and the expression levels were higher in outer (epidermis/exodermis/sclerenchyma) and inner cells (endodermis/pericycle/stele) than cortex cells in the basal zones of roots (10–30 mm from the root-tips) in 10-d-old seedling. The gene expression levels of OsADH1 and OsADH2 in roots after transplantation into paddy fields (before heading) were higher than those before transplantation and close to those after anaerobic treatment (Supplemental Figure S7). However, the gene expression levels of OsADH1 and OsADH2 were dramatically reduced around heading time (Figure 5A;  Supplemental Figure S7). Furthermore, the reduction in OsADH1 transcripts was more severe than that in OsADH2 transcripts (Figure 5B). These expression patterns were similar between WT and las3. Similar to seedlings before transplanting, the expression level of OsADH3 was very low or hardly detected. These data suggest that, of the ADH paralogues, OsADH2 is the most abundant and plays a predominant role in this stage.

Figure 5.

Figure 5

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Expression changes of OsADH1 and OsADH2 after transplanting. (A, B) mRNA levels of OsADH1 and OsADH2 in roots of WT and las3 plants. Copy numbers of the cDNAs synthesized from 1 ng of total RNA are shown. RT-qPCR analyses of three biological replicates. Means ± SDs. *P < 0.05 (t test).

Regulation of OsLsi1 and OsLsi2 by intracellular pH

Lack of oxygen induces a decrease in ATP synthesis efficiency and induces lactate fermentation from pyruvate (Dennis et al., 2000). Furthermore, lactate accumulation, deactivation of proton pumps by lack of ATP, and other metabolism induce cytosolic pH decreases (Dennis et al., 2000). ADH-mediated ethanol fermentation from pyruvate largely moderates cytosolic pH decreases (Ishizawa, 2014). Therefore, a representative effect of ADH deficiency is a cytosolic pH decrease. To investigate whether a change in cytosolic pH affects the expression of OsLsi1 and OsLsi2, roots of WT seedlings were treated with erythrosine B, which is a proton pump inhibitor that decreases cytosolic pH (Lapous et al., 1998; Zhang et al., 2005), and gene expression analysis was performed. Erythrosine B treatment dramatically reduced the expression levels of OsLsi1 and OsLsi2 (Supplemental Figure S8).

To examine the acidification of las3 root cells in hypoxia, we analyzed the intracellular pH using the 31P-nuclear magnetic resonance (31P-NMR) method. Because the 31P chemical shift of Pi compounds is strongly dependent on pH, 31P-NMR can be applied to estimate the intracellular pH in plant root cells. The plants were continuously cultured in a stagnant agar solution, which mimics anaerobic paddy conditions, until heading. Development of root aerenchyma was observed, indicating that the stagnant treatment mimicked the anaerobic condition (Figure 6A). Previous studies using 31P-NMR have shown that three peaks consisting of glucose-6-P, cytosolic inorganic Pi, and vacuolar Pi were obtained from root-tips of maize (Roberts et al., 1981) or mung bean (Torimitsu et al., 1984). For our 31P-NMR measurement, one broad peak with a distinct difference in the 31P chemical shift was obtained from the roots of WT and las3 (Figure 6B). The pH difference between WT and las3 was estimated to be ∼0.6 units based on a relationship between the chemical shift of Pi and pH (Supplemental Figure S9), indicating that the intracellular pH of las3 roots was lower than WT roots. Although we could not separate the cytosolic Pi from the broad peak, intracellular acidification of las3 roots was certainly observed.

Figure 6.

Figure 6

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Intercellular pH measurements in roots by 31P-NMR and a possible mechanism for the low-As phenotype of the las3 mutant. (A) Morphology of plants grown in a stagnant deoxygenated nutrient solution. After heading, cross-sections of 10-mm root tips were prepared using a microtome. (B) 31P-NMR spectra of hypoxic roots. 20–30-mm pieces of root tips were used for the 31P-NMR analysis. MDP was used as an external reference (17.7 ppm). Two spectra were magnified to show the difference in peak positions between WT and las3. Representative data for three replicates are shown. (C) Provisional model of the mechanism for the low-As phenotype in las3. The diagram shows root cells in WT and las3 plants at the heading stage. Under anaerobic conditions, OsADH2 effectively moderates cytosolic pH decreases by accelerating ethanol fermentation in WT (left panel). In the las3 mutant, a decrease in ethanol fermentation by mutation facilitates a decrease in cytosolic pH. The cytosolic pH decrease suppresses the gene expression of OsLsi1 and OsLsi2 and reduces the activity of OsLsi2 protein, which is driven by the proton gradient, and thereby suppresses the transfer of As via transmembrane pathways.

To examine a possible factor of intracellular acidification of las3, the lactate levels of roots were measured after anaerobic treatments. A difference in lactate levels in young roots was not observed between WT and las3. Similarly, a significant difference was not found in roots of headed plants, although las3 showed slightly higher levels than WT (Supplemental Figure S10).

Discussion

Using a forward genetic approach, we isolated the low-As mutant las3 and discovered that deficiency in OsADH2 reduces uptake of As, probably due to suppression of silicate transporters.

ADHs exist in many organisms (such as bacteria, yeast, animals, and plants), and a number have been well characterized (de Smidt et al., 2008; Raj et al., 2014). In plants, some ADHs catalyze the conversion of acetaldehyde to ethanol and ethanol to acetaldehyde as a part of fermentation (Strommer, 2011). Involvement of ADH in As uptake has not been reported. Although disruption mutants of OsADH2 were generated in a previous study (Terada et al., 2007), the mutant phenotypes and physiological roles of OsADH2 remained unclear. In this study, a forward genetic approach identified the involvement of OsADH2 in As uptake. Characterization of las3 provided valuable insight about rice ADHs.

ADH paralogues are thought to have similar enzymatic properties, and their tissue specificities overlap to some degree. Gene expression and immunoblot analyses indicated that OsADH2 most efficiently accumulated in root tissue under anaerobic conditions (Figure 3). Generally, differences in protein levels are due to differences in translation efficiency or protein stability. Since the 5′UTR of OsADH2 is a translation enhancer (Sugio et al., 2008), the higher translation efficiency of OsADH2 is suggested to be a major cause of the difference in protein levels. The total ADH level markedly decreased in las3 compared with WT, although the transcript levels of ADHs were similar (Figure 3C). This was probably due to reduced protein stability caused by disruption of the structural zinc-binding site.

The low-As phenotype of las3 began to be expressed toward heading time and was not observed in young plants before heading under our experimental conditions (Figure 4A;  Supplemental Figure S1). The expression levels of OsADH1 and OsADH2 in roots were suppressed with maturity (Figure 5A). Particularly, the expression level of OsADH1 was lower than OsADH2 during heading (Figure 5B). The particularly high dependency of OsADH2 on the anaerobic response in this period can be expected. Therefore, it is likely that the low-As phenotype of OsADH2-deficient mutants appeared in this period. Conversely, the functions of the ADH paralogues in young plant roots may be redundant in many cases. In contrast to plants in the heading stage, young plants can express high amounts of ADHs in response to anaerobic treatment (Supplemental Figure S7), and thereby anaerobic metabolism could be maintained by other ADH paralogues (probably OsADH1) in young las3 plants.

In plants, a representative role of ADH is catalysis of ethanol fermentation, and dysfunction of ADH facilitates acidification of the cytosol. In addition, a previous study suggested that transport of silicate by OsLsi2 is an energy-dependent, active process that is driven by the proton gradient (Ma et al., 2011), presuming that a cytosolic pH decrease could suppress the activity of the OsLsi2 protein. Therefore, we suspected that silicate transporter suppression induced by a cytosolic pH decrease was a cause of the low-As phenotype. We found that erythrosine B treatment of the WT plant markedly reduced the expression levels of OsLsi1 and OsLsi2 (Supplemental Figure S8), although it is possible that some effect of erythrosine B other than a cytosolic pH decrease caused the negative regulation of OsLsi1 and OsLsi2. To prove our hypotheses, direct measurements of cytosolic pH would be effective but are technically difficult. Although challenging, we applied a 31P-NMR method to estimate the intracellular pH of roots of headed plants grown in the hypoxic solution. Unexpectedly, the spectrum showed one broad peak for both WT and las3 (Figure 6B). A similar result was observed in the 31P-NMR spectrum of mung bean young roots under anaerobic conditions (Torimitsu et al., 1984). Some reasons for the appearance of the broad peak are considered. The peaks of cytosolic Pi and vacuolar Pi were fused, probably due to a reduction in pH difference between the cytosol and vacuole in hypoxic roots (Roberts et al., 1984; Torimitsu et al., 1984). Moreover, the development of aerenchyma associated with hypoxia (Figure 6A) and large vacuole formation in mature roots make it more difficult to identify Pi in the cytosol separately from in the vacuole. Therefore, our results indicate that the intracellular pH combined the cytosolic pH and the vacuolar pH, and that the vacuolar pH probably contributes greatly because the pH values for both plant roots were estimated to be ˂6.0 (Figure 6B;  Supplemental Figure S9). Nevertheless, the 31P-NMR spectra showed the intracellular acidification of las3 roots under anaerobic conditions. Based on this circumstantial evidence, we proposed a provisional model of the mechanism for the low-As phenotype in the las3 mutant, as shown in Figure 6C. However, intracellular acidification of las3 roots did not correspond with changes in lactate levels in roots because a significant increase was not found (Supplemental Figure S10). In addition, it is unclear whether the 0.6 unit decrease in intracellular pH of rice roots was responsible for the suppression of OsLsi1 and OsLsi2. Therefore, further multidirectional approaches would be required to reveal the events that are responsible for the low-As phenotype of las3. For instance, identification of factors that mediate gene regulation of OsLsi1 and OsLsi2 would not only shed light on such events but also enable the development of alternative ways to limit silicate transporters without a deficiency of ADH.

One of the major purposes of this study is to provide candidate breeding material for reducing As levels in rice. Although OsLsi1 and OsLsi2 disruptants showed a significant reduction in As accumulation, the grain yields were only 10% for lsi1 and 40% for lsi2 compared with WT (Ma et al., 2006, 2007). However, the OsADH2 disruptant (las3) maintained >90% grain yield relative to that of WT. This difference may be attributed to the shoot Si concentration: the concentration was ˂1% for lsi1, ∼1% for lsi2, and 3% for las3, relative to ∼4–5% for WT plants. Because the limited function of silicate transporters in las3 occurs during specific times, the negative effect of a lack of silicate is supposed to be moderate. Additionally, we suspected that the expression of the low-As trait of las3 is sensitive to water control in paddy fields. However, the trait was also observed in paddy fields under intermittent irrigation and water-saving irrigation (Figure 1A), demonstrating that the requirements for expression of the low-As trait are not strict. Under our experimental conditions, the decreasing rate of As in las3 was ∼30% for grains and >50% for straws compared with those of WT when plants were cultivated under the same water regimes (Figure 1A). Moreover, growing las3 under aerobic water-saving conditions would reduce As concentration by ∼60% for grains and 90% for straws compared with those of WT grown under anaerobic flooding conditions. Thus, the las3 mutant would be a potential breeding material for the development of low-As rice cultivars.

Materials and methods

Plant materials and growth conditions

The japonica rice cultivar “Koshihikari” (O. sativa L.) was used as WT in this study unless otherwise noted. “Koshihikari” was mutagenized with carbon ions (Ishikawa et al., 2012). Based on the grain As concentration, las3 was obtained from M2 plants (∼3000) grown in pots filled with paddy soil. A Tos17 insertion line of “Nipponbare” was obtained from the Rice Genome Research Center, NARO. The ADH2 mutant line, having G207D in the “Koshihikari” genetic background, was developed by treating single zygotic cells with N-methyl-N-nitroso urea (MNU) using the modified TILLING system (Suzuki et al., 2008). The primers for TILLING are listed in Supplemental Table S2.

The low-As phenotype and agronomic traits of las3 were examined in a paddy field under three water regimes: flooded irrigation, intermittent irrigation, and water-saving conditions (Ishikawa et al., 2016). The plants grown under flooded conditions were harvested at 5 weeks (before heading), 11 weeks (heading), and 14 or 16 weeks (after heading) after transplanting to analyze the minerals in aerial parts and gene expression in roots. For plants grown under flooded conditions, aerial parts were also divided into grains, husks, leaf blades, leaf sheaths, nodes, and internodes at each nodal position. The agronomic traits of las3 and WT were evaluated according to the methods of Abe et al. (2017).

To evaluate low-As phenotypes for allelic mutants of las3, the Tos17 insertion line, las3, and the WT cultivars “Nipponbare” and “Koshihikari” were cultivated in pots filled with paddy soil containing a relatively high As concentration (8 mg kg−1) under continuously flooded conditions. Moreover, the MNU mutant line, the WT cultivar “Koshihikari,” and las3 were also cultivated in a paddy field containing a relatively low As concentration (1.4 mg kg−1) under continuously flooded conditions. After harvesting grains and straws from each sample, the As concentrations were analyzed as described below.

To compare As uptake by roots and subsequent transfer to shoots between WT and las3, two-week-old seedlings grown in a half-strength Kimura B solution (pH 5.0) were treated for 3 d with varying levels of As(III) in the nutrient solution in a Biotron with a 16-h light (28°C)/8-h dark (25°C) cycle.

For plant exposure to anaerobic conditions, seedlings were grown on Murashige and Skoog (MS) medium (MS salt mix, B5 vitamin, pH 5.8, and 0.25% w/v Gelrite) at 32°C using a 16-h light/8-h dark cycle and then transferred into water or liquid MS medium in a shaded airtight box from which oxygen was removed by the AnaeroPack system (Mitsubishi Gas Chemical Co., Tokyo, Japan). In the anaerobic treatment shown in Figure 3 and Supplemental Figure S7, 7-d-old seedlings grown on solid MS medium were immediately transferred into liquid MS medium. In the case of Figure 3, B and C, to reduce the background level of the hypoxic response, 6-d-old seedlings were incubated on wet filter paper for 1 d (aerobic treatment) before anaerobic treatment. For erythrosine B treatment, 7-d-old seedlings were incubated in liquid MS medium containing 0.1 mM erythrosine B.

31P-NMR spectroscopy

To examine the intracellular pH in roots during the heading stage, WT and las3 plants were continuously exposed to a stagnant deoxygenated nutrient solution. The stagnant solution contained 0.1% (w/v) agar and 2 mM MES–Tris (pH 5.8) in full-strength Kimura B solution. The solution was flushed with nitrogen gas, and the dissolved oxygen level was decreased to be ˂1.0 mg L−1. The solution was renewed every 7 d and the pH adjusted daily. After the first panicle emerged, each plant was treated for 3 d with 10 mM P in the stagnant solution, after which it was cultured in the stagnant solution without P for 12 h to remove excess P from water-free space in the roots. Because Lsi1 and Lsi2 were highly expressed in the basal zone, a 20–30-mm portion from the root tips was excised and transferred to a 5-mm NMR tube.

31P-NMR spectra were obtained using an ECA600 FT NMR (JEOL, Tokyo, Japan) at 243 MHz. Acquisition parameters for NMR analyses were as follows: 30° pulse width of 4.24 µs, acquisition time of 0.304 s, pulse delay of 2 s, and broadband proton decoupling at room temperature. The total accumulation time was 3 h. A broadening factor of 2.00 Hz was used for Fourier transformation. Chemical shifts (ppm) for P in root samples were determined with respect to the external standard, 10 mM of methylene diphosphonate (MDP; 17.7 ppm) dissolved in D2O for external locking. A capillary containing the external standard solution was inserted in the 5-mm NMR tube with root samples. In addition, the pH dependence of the chemical shift of Pi was investigated using orthophosphate solution adjusted to pH 4.5–8 by mixing different proportions of 5 mM KH2PO4 and K2HPO4 solutions with 100 mM KC1 and 5 mM MgSO4.

Elemental analyses

Total As concentrations in plant samples were analyzed according to the method of Ishikawa et al. (2016). Briefly, the samples were digested with a 5:1 (v/v) mixture of 60% (w/w) HNO3- 30% (w/w) H2O2 at 105°C for 4 h in a block heater and then diluted with Milli-Q water and filtered through disposable syringe filters. Concentrations of As were determined by inductively coupled plasma-mass spectrometry (ICP-MS; ELAN DRC-e, Perkin Elmer, Inc., Waltham, MA, USA). The same digested solution was used for P analysis by inductively coupled plasma optical emission spectrometry (ICP-OES; Agilent Technologies, Santa Clara, CA, USA). For Si analysis, the dried samples were digested with a 7:2:1 (v/v/v) mixture of 60% (w/w) HNO3- 30% (w/w) H2O2- 50% (w/w) hydrofluoric acid (HF) in a microwave oven (Ethos TC, Milestone, Sorisole, Italy). After cooling, 6 mL of saturated boric acid was added to each digested sample to block the interference of HF and then heated again in a microwave oven. The Si concentrations of the samples were analyzed by ICP-OES.

Identification of the las3 locus

Genetic linkage analysis and whole-genome resequencing were carried out for identification of the las3 locus. The details of these procedures were essentially the same as those previously reported (Hayashi et al., 2017). For genetic linkage analysis, the isolated las3 mutant (“Koshihikari” background) was crossed with the indica rice cultivar “Habataki.” The genomic DNA of F2 plants was analyzed by using an SNP array consisting of 768 SNPs from a world core SNP set according to the method reported by Kuramata et al. (2013) and Yonemaru et al. (2014). The concentrations of As in the plant tissues (grains, husks, and straws) harvested from F2 plants grown in paddy soil were determined by ICP-MS. Whole-genome resequencing of the WT “Koshihikari” and the las3 mutant was performed according to the methods of Takagi et al. (2013) and Hayashi et al. (2017).

Gene expression analyses

Total RNA was extracted from roots using an RNeasy Mini Kit (Qiagen, Venlo, Netherlands). First-strand cDNA was synthesized from total RNA using Rever-Tra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). Reverse transcription-quantitative PCR (RT-qPCR) was performed using SYBR Premix Ex Taq (Takara Bio Inc., Kusatsu, Shiga, Japan) or KOD SYBR qPCR Mix (Toyobo). To calculate relative mRNA levels, the 25S rRNA gene was used as an internal reference. The copy numbers of OsADH1, OsADH2, and OsADH3 transcripts in the RT products were calculated by generating a standard curve using a plasmid harboring OsADH1, OsADH2, and OsADH3 cDNA.

Generation of disruption mutants

Disruption mutants of OsADH1 and OsADH3 were generated by genome editing using the CRISPR/Cas9 system for rice (Endo et al., 2019). Transgenic rice plants were generated by Agrobacterium (Rhizobium radiobacter strain EHA105)-mediated transformation using calli induced from “Koshihikari” seeds. Genomic DNA was extracted from T0 or T1 plants, and disruption mutants were selected by sequencing analyses. Plants were cultivated in pots filled with paddy soil containing 8 mg kg−1 As under continuously flooded conditions, and As concentrations in grains were analyzed by ICP-MS.

Immunoblot analysis

The VNPKDHSKPVH peptide derived from OsADH2 was synthesized and used to raise a polyclonal antibody in rabbit (Scrum Inc., Tokyo, Japan). This antibody reacts with OsADH1, OsADH2, and OsADH3 almost equally (Supplemental Figure S5). Frozen roots were ground to a fine powder and homogenized in extraction buffer (50 mM Tris–HCl pH 6.8, 8 M urea, 4% w/v Sodium dodecyl sulfate(SDS) , 20% v/v glycerol, 5% v/v 2-mercaptoethanol, and 0.01% w/v bromophenol blue). The mixtures were centrifuged at 20,000 g for 5 min, and the supernatants were used as total protein samples. The total proteins were subjected to immunoblot analysis using the polyclonal rabbit antibody and an anti-rabbit Ig antibody conjugated to horseradish peroxidase after electrophoresis on 10% SDS-PAGE.

Lactate measurement

Lactate in roots of 7-d-old seedlings or plants with panicles grown under anaerobic conditions was extracted with 5% (v/v) HClO4. After centrifugation (20,000 g, 5 min, 4°C), the supernatant was neutralized with KOH. L-lactate concentrations (µmol g−1 FW) were determined with an L-lactic acid determination kit (No. 10139084035; Roche, Mannheim, Germany).

Oligonucleotides

The oligonucleotides used in this study are listed in Supplemental Table S2.

Statistics

Data were analyzed by unpaired t test with F test or Tukey’s multiple comparison test after one-way ANOVA.

Accession numbers

The data sets obtained from next-generation sequencing in the las3 (DRA010468) and the nucleotide sequence of OsADH2 in the las3 (LC557141) have been deposited in the DNA Data Bank of Japan (https://www.ddbj.nig.ac.jp/). In addition, sequence data from this article can be found in the Rice Annotation Project Database (https://rapdb.dna.affrc.go.jp/) under the following accession numbers: Os11g0210300 (OsADH1), Os11g0210500 (OsADH2), Os11g0210600 (OsADH3), Os02g0745100 (OsLsi1), and Os03g0107300 (Lsi2).

Supplemental data

The following supplemental materials are available in the online version of this article.

Supplemental Figure S1. As contents in young roots and shoots of WT and las3.

Supplemental Figure S2. Detailed information about the identification of the las3 mutation.

Supplemental Figure S3. Amino acid sequences of rice ADH paralogues.

Supplemental Figure S4. Information on the osadh1 and osadh3 mutants.

Supplemental Figure S5. Evaluation of anti-ADH antibody.

Supplemental Figure S6. P uptake after transplanting.

Supplemental Figure S7. Comparison of mRNA levels of OsADH1 and OsADH2 between young plants and transplanted plants.

Supplemental Figure S8. mRNA levels of OsLsi1 and OsLsi2 in roots of WT and las3 plants after treatment with erythrosine B.

Supplemental Figure S9. Relationship between the chemical shifts of Pi and pH by 31P-NMR analysis.

Supplemental Figure S10. Lactate levels in young and mature roots after anaerobic treatment.

Supplemental Table S1. Agronomic traits of las3.

Supplemental Table S2. Oligonucleotides used in this study.

Supplementary Material

kiab086_Supplementary_Data
Click here for additional data file. (781.3KB, pdf)

Acknowledgments

The use of the SNP array was supported by a genome-support grant from the National Institute of Agrobiological Sciences (current institute: Institute of Crop Science, NARO). The 31P-NMR experiment was conducted at an advanced analysis center, NARO. We thank Dr. M. Nakazono (University of Tokyo) for providing the rad1 mutant seeds. We also thank Mr. H. Yamaguchi and Mr. T. Kamada (Tsukuba Technical Support Center, NARO) for expert field assistance and Ms. A. Arai, Ms. M, Chiba, Ms. Y. Yamanaka, Ms. H. Sakurai, Ms. K. Kojima, Ms. K. Ms. Goto, Ms. K. Kawashima, Ms. T. Omura, and Ms. A. Hikono (Institute for Agro-Environmental Sciences, NARO) for their laboratory assistance.

Funding

This work was supported by a Grant-in-Aid for Scientific Research (KAKENHI Grant Number JP18K05380) from Japan Society for the Promotion of Science (JSPS) and a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics-based Technology for Agricultural Improvement, LCT-0008). 

Conflict of interest statement. Authors have no conflict of interest.

 

S.H. and S.I. designed the research; S.H., M.K., T.A, M.I., and S.I. performed the genetic and molecular experiments; T.A. and S.I. performed the field experiments; H.T. and S.I. performed metal analyses; N.Y. and S.I. performed the 31P-NMR experiment; H.T. performed whole-genome sequencing; K.S. and S.I produced rice mutant materials; S.H. and S.I. wrote the manuscript. All authors approved the final version of the manuscript.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is: Satoru Ishikawa (isatoru@affrc.go.jp).

References

  1. Abe T, Ito M, Takahashi R, Honma T, Sekiya N, Shirao K, Kuramata M, Murakami M, Ishikawa S (2017) Breeding of a practical rice line ‘TJTT8’ for phytoextraction of cadmium contamination in paddy fields. Soil Sci Plant Nutr  63: 388–395 [Google Scholar]
  2. Akahane I, Suda A, Ishikawa S, Abe T, Baba K, Furuya M, Yamaguchi N (2020) Distribution dynamics of arsenic and silicon in different parts of rice grown under field conditions. Soil Sci Plant Nutr  66:784–792 DOI:10.1080/00380768.2020.1807884 [Google Scholar]
  3. Arao T, Kawasaki A, Baba K, Mori S, Matsumoto S (2009) Effects of water management on cadmium and arsenic accumulation and dimethylarsinic acid concentrations in Japanese rice. Environ Sci Technol  43: 9361–9367 [DOI] [PubMed] [Google Scholar]
  4. Catarecha P, Segura MD, Franco-Zorrilla JM, Garcia-Ponce B, Lanza M, Solano R, Paz-Ares J, Leyva A (2007) A mutant of the Arabidopsis phosphate transporter PHT1;1 displays enhanced arsenic accumulation. Plant Cell  19: 1123–1133 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. de Smidt O, du Preez JC, Albertyn J (2008) The alcohol dehydrogenases of Saccharomyces cerevisiae: a comprehensive review. Fems Yeast Res  8: 967–978 [DOI] [PubMed] [Google Scholar]
  6. Delaney ML, Onderdonk AB (1997) Evaluation of the anaeropack system for growth of clinically significant anaerobes. J Clin Microbiol  35: 558–562 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dennis ES, Dolferus R, Ellis M, Rahman M, Wu Y, Hoeren FU, Grover A, Ismond KP, Good AG, Peacock WJ (2000) Molecular strategies for improving waterlogging tolerance in plants. J Exp Bot  51: 89–97 [PubMed] [Google Scholar]
  8. Endo M, Mikami M, Endo A, Kaya H, Itoh T, Nishimasu H, Nureki O, Toki S (2019) Genome editing in plants by engineered CRISPR-Cas9 recognizing NG PAM. Nat Plants  5: 14–17 [DOI] [PubMed] [Google Scholar]
  9. Enstone DE, Peterson CA, Ma FS (2002) Root endodermis and exodermis: structure, function, and responses to the environment. J Plant Growth Regul  21: 335–351 [Google Scholar]
  10. Hayashi S, Kuramata M, Abe T, Takagi H, Ozawa K, Ishikawa S (2017) Phytochelatin synthase OsPCS1 plays a crucial role in reducing arsenic levels in rice grains. Plant J  91: 840–848 [DOI] [PubMed] [Google Scholar]
  11. Honma T, Ohba H, Kaneko-Kadokura A, Makino T, Nakamura K, Katou H (2016a) Optimal soil Eh, pH, and water management for simultaneously minimizing arsenic and cadmium concentrations in rice grains. Environ Sci Technol  50: 4178–4185 [DOI] [PubMed] [Google Scholar]
  12. Honma T, Ohba H, Kaneko A, Nakamura K, Makino T, Katou H (2016b) Effects of soil amendments on arsenic and cadmium uptake by rice plants (Oryza sativa L. cv Koshihikari) under different water management practices. Soil Sci Plant Nutr  62: 349–356 [Google Scholar]
  13. Hughes MF (2002) Arsenic toxicity and potential mechanisms of action. Toxicol Lett  133: 1–16 [DOI] [PubMed] [Google Scholar]
  14. Ishikawa S, Arao T, Makino T (2019) Agronomic strategies for reducing arsenic risk in rice. In H Yamauchi and G Sun, eds, Arsenic Contamination in Asia. Current Topics in Environmental Health and Preventive Medicine. Springer, Singapore, pp 181–198 [Google Scholar]
  15. Ishikawa S, Ishimaru Y, Igura M, Kuramata M, Abe T, Senoura T, Hase Y, Arao T, Nishizawa NK, Nakanishi H (2012) Ion-beam irradiation, gene identification, and marker-assisted breeding in the development of low-cadmium rice. Proc Natl Acad Sci USA  109: 19166–19171 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ishikawa S, Makino T, Ito M, Harada K, Nakada H, Nishida I, Nishimura M, Tokunaga T, Shirao K, Yoshizawa C  et al. (2016) Low-cadmium rice (Oryza sativa L.) cultivar can simultaneously reduce arsenic and cadmium concentrations in rice grains. Soil Sci Plant Nutr  62: 327–339 [Google Scholar]
  17. Ishizawa K (2014) Intracellular pH regulation of plant cells under anaerobic conditions. In van Dongen J., Licausi F., eds, Low-Oxygen Stress in Plants. Plant Cell Monographs, Vol 21. Springer, Vienna, pp 59– 74 [Google Scholar]
  18. Kumarathilaka P, Seneweera S, Ok YS, Meharg AA, Bundschuh J (2020) Mitigation of arsenic accumulation in rice: an agronomical, physico-chemical, and biological approach - A critical review. Crit Rev Environ Sci Technol  50: 31–71 [Google Scholar]
  19. Kuramata M, Abe T, Kawasaki A, Ebana K, Shibaya T, Yano M, Ishikawa S (2013) Genetic diversity of arsenic accumulation in rice and QTL analysis of methylated arsenic in rice grains. Rice  6: 3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lapous D, Mathieu Y, Guern J, Lauriere C (1998) Increase of defense gene transcripts by cytoplasmic acidification in tobacco cell suspensions. Planta  205: 452–458 [Google Scholar]
  21. Ma JF, Nishimura K, Takahashi E (1989) Effect of silicon on the growth of rice plant at different growth-stages. Soil Sci Plant Nutr  35: 347–356 [Google Scholar]
  22. Ma JF, Tamai K, Yamaji N, Mitani N, Konishi S, Katsuhara M, Ishiguro M, Murata Y, Yano M (2006) A silicon transporter in rice. Nature  440: 688–691 [DOI] [PubMed] [Google Scholar]
  23. Ma JF, Yamaji N (2006) Silicon uptake and accumulation in higher plants. Trends Plant Sci  11: 392–397 [DOI] [PubMed] [Google Scholar]
  24. Ma JF, Yamaji N (2008) Functions and transport of silicon in plants. Cell Mol Life Sci  65: 3049–3057 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ma JF, Yamaji N, Mitani-Ueno N (2011) Transport of silicon from roots to panicles in plants. Proc Jpn Acd Ser B  87:377–385 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ma JF, Yamaji N, Mitani N, Tamai K, Konishi S, Fujiwara T, Katsuhara M, Yano M (2007) An efflux transporter of silicon in rice. Nature  448: 209–212 [DOI] [PubMed] [Google Scholar]
  27. Ma JF, Yamaji N, Mitani N, Xu XY, Su YH, McGrath SP, Zhao FJ (2008) Transporters of arsenite in rice and their role in arsenic accumulation in rice grain. Proc Natl Acad Sci USA  105: 9931–9935 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Magonet E, Hayen P, Delforge D, Delaive E, Remacle J (1992) Importance of the structural zinc atom for the stability of yeast alcohol-dehydrogenase. Biochem J  287: 361–365 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Makino T, Nakamura K, Katou H, Ishikawa S, Ito M, Honma T, Miyazaki N, Takehisa K, Sano S, Matsumoto S  et al. (2016) Simultaneous decrease of arsenic and cadmium in rice (Oryza sativa L.) plants cultivated under submerged field conditions by the application of iron-bearing materials. Soil Sci Plant Nutr  62: 340–348 [Google Scholar]
  30. Matsumura H, Takano T, Yoshida KT, Takeda G (1995) A rice mutant lacking alcohol-dehydrogenase. Breed Sci  45: 365–367 [Google Scholar]
  31. Oguri T, Yoshinaga J, Tao H, Nakazato T (2014) Inorganic arsenic in the Japanese diet: daily intake and source. Arch Environ Contam Toxicol  66: 100–112 [DOI] [PubMed] [Google Scholar]
  32. Raj SB, Ramaswamy S, Plapp BV (2014) Yeast alcohol dehydrogenase structure and catalysis. Biochem  53: 5791–5803 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Roberts JKM, Callis J, Wemmer D, Walbot V, Jardetzky O (1984) Mechanism of cytoplasmic pH regulation in hypoxic maize root-tips and its role in survival under hypoxia. Proc Natl Acad Sci USA  81: 3379–3383 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Roberts JKM, Wadejardetzky N, Jardetzky O (1981) Intracellular pH measurements by 31P nuclear magnetic-resonance influence of factors other than pH on 31P chemical-shifts. Biochem  20: 5389–5394 [DOI] [PubMed] [Google Scholar]
  35. Roberts JKM, Wemmer D, Ray PM, Jardetzky O (1982) Regulation of cytoplasmic and vacuolar pH in maize root-tips under different experimental conditions. Plant Physiol  69: 1344–1347 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Saika H, Matsumura H, Takano T, Tsutsumi N, Nakazono M (2006) A point mutation of Adh1 gene is involved in the repression of coleoptile elongation under submergence in rice. Breed Sci  56: 69–74 [Google Scholar]
  37. Schoof RA, Yost LJ, Eickhoff J, Crecelius EA, Cragin DW, Meacher DM, Menzel DB (1999) A market basket survey of inorganic arsenic in food. Food Chem Toxicol  37: 839–846 [DOI] [PubMed] [Google Scholar]
  38. Strommer J (2011) The plant ADH gene family. Plant J  66: 128–142 [DOI] [PubMed] [Google Scholar]
  39. Sugio T, Satoh J, Matsuura H, Shinmyo A, Kato K (2008) The 5′- untranslated region of the Oryza sativa alcohol dehydrogenase gene functions as a translational enhancer in monocotyledonous plant cells. J Biosci Bioeng  105: 300–302 [DOI] [PubMed] [Google Scholar]
  40. Suzuki T, Eiguchi M, Kumamaru T, Satoh H, Matsusaka H, Moriguchi K, Nagato Y, Kurata N (2008) MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice. Mol Genet Genomics  279: 213–223 [DOI] [PubMed] [Google Scholar]
  41. Takagi H, Uemura A, Yaegashi H, Tamiru M, Abe A, Mitsuoka C, Utsushi H, Natsume S, Kanzaki H, Matsumura H  et al. (2013) MutMap-Gap: whole-genome resequencing of mutant F2 progeny bulk combined with de novo assembly of gap regions identifies the rice blast resistance gene Pii. New Phytol  200: 276–283 [DOI] [PubMed] [Google Scholar]
  42. Takahashi Y, Minamikawa R, Hattori KH, Kurishima K, Kihou N, Yuita K (2004) Arsenic behavior in paddy fields during the cycle of flooded and non-flooded periods. Environ Sci Technol  38: 1038–1044 [DOI] [PubMed] [Google Scholar]
  43. Terada R, Johzuka-Hisatomi Y, Saitoh M, Asao H, Iida S (2007) Gene targeting by homologous recombination as a biotechnological tool for rice functional genomics. Plant Physiol  144: 846–856 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Torimitsu K, Yazaki Y, Nagasuka K, Ohta E, Sakata M (1984) Effect of external pH on the cytoplasmic and vacuolar pHs in mung bean root-tip cells - A 31P nuclear magnetic-resonance study. Plant Cell Physiol  25: 1403–1409 [Google Scholar]
  45. World Health Organization. (2011) Arsenic in drinking-water. Background Document for Development of WHO Guidelines for Drinking-water Quality. http://www.who.int/water_sanitation_health/publications/arsenic/en/
  46. Wu C, Ye ZH, Li H, Wu SC, Deng D, Zhu YG, Wong MH (2012) Do radial oxygen loss and external aeration affect iron plaque formation and arsenic accumulation and speciation in rice?  J Exp Bot  63: 2961–2970 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Xie Y, Wu R (1989) Rice alcohol-dehydrogenase genes - anaerobic induction, organ specific expression and characterization of Cdna clones. Plant Mol Biol  13: 53–68 [DOI] [PubMed] [Google Scholar]
  48. Yamaji N, Ma JF (2007) Spatial distribution and temporal variation of the rice silicon transporter Lsi1. Plant Physiol  143: 1306–1313 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yamaji N, Ma JF (2011) Further characterization of a rice silicon efflux transporter, Lsi2. Soil Sci Plant Nutr  57: 259–264 [Google Scholar]
  50. Yonemaru J, Mizobuchi R, Kato H, Yamamoto T, Yamamoto E, Matsubara K, Hirabayashi H, Takeuchi Y, Tsunematsu H, Ishii T  et al. (2014) Genomic regions involved in yield potential detected by genome-wide association analysis in Japanese high-yielding rice cultivars. BMC Genomics  15: 346 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhang ZS, Ramirez J, Reboutier D, Brault M, Trouverie J,, Pennarun AM, Amiar Z, Biligui B, Galagovsky L, Rona JP (2005) Brassinosteroids regulate plasma membrane anion channels in addition to proton pumps during expansion of Arabidopsis thaliana cells. Plant Cell Physiol  46: 1494–1504 [DOI] [PubMed] [Google Scholar]
  52. Zhao FJ, Wang P (2020) Arsenic and cadmium accumulation in rice and mitigation strategies. Plant Soil  446: 1–21 [Google Scholar]

Associated Data

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Supplementary Materials

kiab086_Supplementary_Data
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