Abstract
The heavy metal cadmium (Cd) is toxic to humans, and its accumulation in rice grains is a major agricultural problem. Rice has seven putative metal transporter NRAMP genes, but microarray analysis showed that only OsNRAMP1 is highly up-regulated by iron (Fe) deficiency. OsNRAMP1 localized to the plasma membrane and transported Cd as well as Fe. OsNRAMP1 expression was observed mainly in roots and was higher in the roots of a high-Cd-accumulating cultivar (Habataki) than in those of a low-Cd-accumulating cultivar (Sasanishiki). The amino acid sequence of OsNRAMP1 in the Sasanishiki and Habataki cultivars was found to be 100% identical. These results suggest that OsNRAMP1 participates in cellular Cd uptake and that the differences observed in Cd accumulation among cultivars are because of differences in OsNRAMP1 expression levels in roots.
Keywords: cadmium, metal transporter, OsNRAMP1, phytoremediation, rice
Cadmium (Cd) is among the most toxic heavy metals in humans and causes serious health problems. As rice is a dietary staple for Asians, the accumulation of Cd in rice grains is an increasingly significant agricultural issue. Although the mechanism underlying the uptake and translocation of Cd in plants is not completely understood, some iron (Fe) transporters such as OsIRT1 and OsIRT2 have been reported to take up Cd as well as Fe.1 Fe deficiency in plants induces expression of Fe transporters that enhance uptake and translocation. As a result, Cd uptake and translocation are also enhanced in rice during Fe acquisition.
The NRAMP (natural resistance-associated macrophage proteins) family of metal transporters has been identified in several plant species,2-7 and many of the NRAMP family proteins function as Fe transporters. In Arabidopsis, AtNRAMP1, AtNRAMP3, and AtNRAMP4 have been reported to be Fe transporters and also mediate Cd transport.8-12 Rice possesses seven putative NRAMP genes.13 Microarray analysis revealed that only one of these, OsNRAMP1, together with OsIRT1 and OsIRT2, is highly upregulated by Fe deficiency (Table 1). The Fe uptake function of OsNRAMP1 has been demonstrated in yeast.8 Growth of a Cd-sensitive yeast mutant expressing OsNRAMP1 was impaired in the presence of Cd, and the Cd content of OsNRAMP1-expressing yeast was higher than that of yeast harboring a control vector.15 Moreover, an OsNRAMP1:GFP fusion protein localized to the plasma membrane in onion epidermal cells.15 These results indicate that OsNRAMP1 participates in cellular Cd uptake.
Table 1. The induction ratios of the expression of OsNRAMPs and OsIRTs.
| Accession number | Gene name | Roots | Shoots |
|---|---|---|---|
| Os07 g0258400 |
OsNRAMP1 |
15.1 |
290.2 |
| Os03 g0208500 |
OsNRAMP2 |
1.3 |
0.9 |
| Os06 g0676000 |
OsNRAMP3 |
1.0 |
1.1 |
| Os02 g0131800 |
OsNRAMP4 |
0.6 |
0.7 |
| Os07 g0257200 |
OsNRAMP5 |
1.9 |
0.8 |
| Os01 g0503400 |
OsNRAMP6 |
1.1 |
1.0 |
| Os12 g0581600 |
OsNRAMP7 |
0.7 |
1.1 |
| Os03 g0667500 |
OsIRT1 |
11.2 |
2.7 |
| Os03 g0667300 | OsIRT2 | 11.9 | 10.9 |
The induction ratio was calculated as the increased or decreased expression level after Fe deficiency for 1 week compared with expression under normal conditions (-Fe/+Fe). For microarray analysis, a rice 44 K oligo-DNA microarray (Agilent Technologies, Tokyo, Japan) containing 43,144 unique 60-mer oligonucleotides was used. Microarray hybridization, scanning, and data analysis were performed as described previously.14 The ratios are the means of two independent replicates.
The expression of OsNRAMP1 has been observed mainly in the roots at the vegetative stage.15 A search of the microarray database RiceXPro16 (ricexpro.dna.affrc.go.jp/) revealed that steady-state expression of OsNRAMP1 is observed in the whole plant, i.e., the leaf blade, root, stem, anther, ovary, embryo, endosperm, and pistil (Fig. 1). In the leaf blade and stem, OsNRAMP1 expression is higher during the reproductive stage (Fig. 1A, C). This suggests that OsNRAMP1 plays a role in Fe uptake and/or translocation from roots to the aerial parts, including rice grains. Therefore, OsNRAMP1 may also participate in Cd uptake and/or transport throughout the rice plant. OsNRAMP1 expression in roots was increased in the presence of 1 µM Cd, as in conditions of Fe deficiency.15 The level of OsNRAMP1 expression was higher in the roots of high-Cd-accumulating indica cultivars such as Habataki, Jarjan, Anjana Dhan, and Cho-ko-koku than in the roots of low-Cd-accumulating japonica cultivars such as Sasanishiki, Nipponbare, and Tsukinohikari.15 The amino acid sequence of OsNRAMP1 was 100% identical among Sasanishiki, Nipponbare, and Habataki. Thus, differences in the expression levels of OsNRAMP1 in roots may be responsible for the observed differences in Cd accumulation among these cultivars.
Figure 1.
(A) Leaf blade: 27, 76, and 125 d after transplantation (DAT); (B) root: 27 and 76 DAT; (C) stem: 83 and 90 DAT; (D) anther: 0.3–0.6, 0.7–1.0, 1.2–1.5, and 1.6–2.0 mm; (E) ovary: 1, 3, 5, and 7 d after flowering (DAF); (F) embryo: 7, 10, 14, 28, and 42 DAF; (G) endosperm: 7, 10, 14, 28, and 42 DAF; (H) pistil: 5–10, 10–14, and 14–18 cm. Normalized signal intensity for OsNRAMP1 was derived from spatiotemporal profiling of various tissues and organs generated through RiceXPro (http://ricexpro.dna.affrc.go.jp/RXP_0003/index.php). Medians are followed by error bars showing maximum and minimum values. n = 3. Samples were collected at noon.
Recently, quantitative trait loci (QTL) for Cd concentration in rice have been identified on chromosome 7.17-19 The gene responsible for the QTL effect has been identified as OsHMA3 in the Cho-ko-koku and Anjana Dhan cultivars,20,21 but the gene in Habataki has not yet been identified. The amino acid at position 80 in OsHMA3 is critical for its transport function; mutation of this amino acid causes a loss of the ability to sequester Cd into vacuoles in root cells, resulting in increased Cd translocation from the roots to the shoots in Anjana Dhan.21 The same mutation has been observed for OsHMA3 in Cho-ko-koku and Jarjan,20,22 but not in Habataki, suggesting that this mutation is not the reason for Cd accumulation in Habataki shoots. The Cd content of root cells is an important factor for the translocation of large amounts of Cd from the roots to the shoots and its accumulation therein (Fig. 2). Higher expression levels of OsNRAMP1 in the roots of indica cultivars were responsible for increased Cd uptake in root cells and the resultant higher Cd concentration in the cytoplasm of root cells of high Cd-accumulating cultivars compared with low Cd-accumulating cultivars. These results indicate that OsNRAMP1 is one of the crucial proteins responsible for higher Cd accumulation in rice.
Figure 2.
Proposed model for the role of OsNRAMP1 in Cd translocation from the root to the shoot. In addition to OsIRT1 and OsIRT2, OsNRAMP1 participates in Cd uptake or transport in rice. OsIRT1 is highly expressed in the epidermis and the cortex surrounding the endodermis in the root, and is considered to function in Fe2+ and Cd uptake from the soil.1,23 OsNRAMP1 is proposed to transport Cd into the cytoplasm in the inner layers of the root following Cd uptake from the rhizosphere by OsIRT1, and to participate in the control of Cd availability for loading into the xylem.
Footnotes
Previously published online: www.landesbioscience.com/journals/psb/article/17587
References
- 1.Nakanishi H, Ogawa I, Ishimaru Y, Mori S, Nishizawa NK. Iron deficiency enhances cadmium uptake and translocation mediated by the Fe2+ transporters OsIRT1 and OsIRT2 in rice. Soil Sci Plant Nutr. 2006;52:464–9. doi: 10.1111/j.1747-0765.2006.00055.x. [DOI] [Google Scholar]
- 2.Bereczky Z, Wang HY, Schubert V, Ganal M, Bauer P. Differential regulation of nramp and irt metal transporter genes in wild type and iron uptake mutants of tomato. J Biol Chem. 2003;278:24697–704. doi: 10.1074/jbc.M301365200. [DOI] [PubMed] [Google Scholar]
- 3.Kaiser BN, Moreau S, Castelli J, Thomson R, Lambert A, Bogliolo S, et al. The soybean NRAMP homologue, GmDMT1, is a symbiotic divalent metal transporter capable of ferrous iron transport. Plant J. 2003;35:295–304. doi: 10.1046/j.1365-313X.2003.01802.x. [DOI] [PubMed] [Google Scholar]
- 4.Mizuno T, Usui K, Horie K, Nosaka S, Mizuno N, Obata H. Cloning of three ZIP/Nramp transporter genes from a Ni hyperaccumulator plant Thlaspi japonicum and their Ni2+-transport abilities. Plant Physiol Biochem. 2005;43:793–801. doi: 10.1016/j.plaphy.2005.07.006. [DOI] [PubMed] [Google Scholar]
- 5.Xiao H, Yin L, Xu X, Li T, Han Z. The iron-regulated transporter, MbNRAMP1, isolated from Malus baccata is involved in Fe, Mn and Cd trafficking. Ann Bot. 2008;102:881–9. doi: 10.1093/aob/mcn178. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Oomen RJFJ, Wu J, Lelievre F, Blanchet S, Richaud P, Barbier-Brygoo H, et al. Functional characterization of NRAMP3 and NRAMP4 from the metal hyperaccumulator Thlaspi caerulescens. New Phytol. 2009;181:637–50. doi: 10.1111/j.1469-8137.2008.02694.x. [DOI] [PubMed] [Google Scholar]
- 7.Wei W, Chai T, Zhang Y, Han L, Xu J, Guan Z. The Thlaspi caerulescens NRAMP homologue TcNRAMP3 is capable of divalent cation transport. Mol Biotechnol. 2009;41:15–21. doi: 10.1007/s12033-008-9088-x. [DOI] [PubMed] [Google Scholar]
- 8.Curie C, Alonso JM, Jean ML, Ecker JR, Briat J-F. Involvement of NRAMP1 from Arabidopsis thaliana in iron transport. Biochem J. 2000;347:749–55. doi: 10.1042/0264-6021:3470749. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Thomine S, Wang R, Ward JM, Crawford NM, Schroeder JI. Cadmium and iron transport by members of a plant metal transporter family in Arabidopsis with homology to Nramp genes. Proc Natl Acad Sci USA. 2000;97:4991–6. doi: 10.1073/pnas.97.9.4991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Thomine S, Lelievre F, Debarbieux E, Schroeder JI, Barbier-Brygoo H. AtNRAMP3, a multispecific vacuolar metal transporter involved in plant responses to iron deficiency. Plant J. 2003;34:685–95. doi: 10.1046/j.1365-313X.2003.01760.x. [DOI] [PubMed] [Google Scholar]
- 11.Lanquar V, Lelievre F, Barbier-Brygoo H, Thomine S. Regulation and function of AtNRAMP4 metal transporter protein. Soil Sci Plant Nutr. 2004;50:1141–50. doi: 10.1080/00380768.2004.10408587. [DOI] [Google Scholar]
- 12.Lanquar V, Lelievre F, Bolte S, Hames C, Alcon C, Neumann D, et al. Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron. EMBO J. 2005;24:4041–51. doi: 10.1038/sj.emboj.7600864. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Narayanan NN, Vasconcelos MW, Grusak MA. Expression profiling of Oryza sativa metal homeostasis genes in different rice cultivars using a cDNA macroarray. Plant Physiol Biochem. 2007;45:277–86. doi: 10.1016/j.plaphy.2007.03.021. [DOI] [PubMed] [Google Scholar]
- 14.Ishimaru Y, Masuda H, Suzuki M, Bashir K, Takahashi M, Nakanishi H, et al. Overexpression of the OsZIP4 zinc transporter confers disarrangement of zinc distribution in rice plants. J Exp Bot. 2007;58:2909–15. doi: 10.1093/jxb/erm147. [DOI] [PubMed] [Google Scholar]
- 15.Takahashi R, Ishimaru Y, Senoura T, Shimo H, Ishikawa S, Arao T, et al. The OsNRAMP1 iron transporter is involved in Cd accumulation in rice. J Exp Bot. 2011 doi: 10.1093/jxb/err136. In press. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Sato Y, Antonio BA, Namiki N, Takehisa H, Minami H, Kamatsuki K, et al. RiceXPro: a platform for monitoring gene expression in japonica rice grown under natural field conditions. Nucleic Acids Res. 2011;39:D1141–8. doi: 10.1093/nar/gkq1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Tezuka K, Miyadate H, Katou K, Kodama I, Matsumoto S, Kawamoto T, et al. A single recessive gene controls cadmium translocation in the cadmium hyperaccumulating rice cultivar Cho-Ko-Koku. Theor Appl Genet. 2010;120:1175–82. doi: 10.1007/s00122-009-1244-6. [DOI] [PubMed] [Google Scholar]
- 18.Ueno D, Koyama E, Kono I, Ando T, Yano M, Ma JF. Identification of a novel major quantitative trait locus controlling distribution of Cd between roots and shoots in rice. Plant Cell Physiol. 2009;50:2223–33. doi: 10.1093/pcp/pcp160. [DOI] [PubMed] [Google Scholar]
- 19.Ishikawa S, Abe T, Kuramata M, Yamaguchi M, Ando T, Yamamoto T, et al. A major quantitative trait locus for increasing cadmium-specific concentration in rice grain is located on the short arm of chromosome 7. J Exp Bot. 2010;61:923–34. doi: 10.1093/jxb/erp360. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Miyadate H, Adachi S, Hiraizumi A, Tezuka K, Nakazawa N, Kawamoto T, et al. OsHMA3, a P1B-type of ATPase affects root-to-shoot cadmium translocation in rice by mediating efflux into vacuoles. New Phytol. 2011;189:190–9. doi: 10.1111/j.1469-8137.2010.03459.x. [DOI] [PubMed] [Google Scholar]
- 21.Ueno D, Yamaji N, Kono I, Huang CF, Ando T, Yano M, et al. Gene limiting cadmium accumulation in rice. Proc Natl Acad Sci USA. 2010;107:16500–5. doi: 10.1073/pnas.1005396107. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Ueno D, Koyama E, Yamaji N, Ma JF. Physiological, genetic, and molecular characterization of a high-Cd-accumulating rice cultivar, Jarjan. J Exp Bot. 2011;62:2265–72. doi: 10.1093/jxb/erq383. [DOI] [PubMed] [Google Scholar]
- 23.Ishimaru Y, Suzuki M, Tsukamoto T, Suzuki K, Nakazono M, Kobayashi T, et al. Rice plants take up iron as an Fe3+-phytosiderophore and as Fe2+ Plant J. 2006;45:335–46. doi: 10.1111/j.1365-313X.2005.02624.x. [DOI] [PubMed] [Google Scholar]