Name / Grain Cd accumulation / Subspecies / Country of Origin / References
Nipponbare / Low / Japonica / Japan / (Kashiwagi et al. 2009; Ueno et al. 2009b)
Koshihikari / Low / Japonica / Japan / (Ishikawa et al. 2005)
Kasalath / Medium / Indica / India / (Ishikawa et al. 2005; Kashiwagi et al. 2009)
BadariDhan / High / Indica / Nepal / (Ueno et al. 2009a)
Habataki / High / Indica / Japan / (Ishikawa et al. 2010; Uraguchi et al. 2009)
Milyang23 / High / Indica cross / Korea / (Arao and Ae 2003; Murakami et al. 2008; Yan et al. 2010);
Table S2: Primers used for real-time RT-PCR
Gene / Forward Primer (5’→3’) / Reverse Primer (5’→3’)
eEF-1α / TTTCACTCTTGGTGTGAAGCAGAT / GACTTCCTTCACGATTTCATCGTAA
OsIRT1 / GATGTGCTTCCACCAGATGTTC / GAAGAAGACGAGCACCGA
OsNRAMP1 / AGGAATGAAGGATGTCTGTAG / AGCATCTTCTGGTGAAAGG
IDEF1 (Kobayashi et al. 2009) / ATGGACGACATGGTGCTCC / CTAGGGATTTGTTGTCTGCT
Table S3: Comparison of soil pore water sampling (centrifuge method) and DGT for measuring available elemental concentrations in soil solution. ‘Lab test’ refers to soil subsamples taken from the experimental pots for use in the bench-top DGT protocol, and also pore water sampling of the prepared soil at water-holding capacity. “b.d.” = below detection limit.
Total soil conc.
(mg kg-1) / Soil pore water -
Lab test
(µg kg-1) / Soil pore water - (A) Aerobic
(µg kg-1) / Soil pore water - (C) Flooded
(µg kg-1) / DGT -
Lab test
(µg kg-1) / DGT - (A) Aerobic
(µg kg-1) / DGT - (C)
Flooded
(µg kg-1)
Cd Spiked soil / Cd / 0.43 / 0.87 / 1.40 / 0.34 / 0.48 / 0.52 / 0.14
Unspiked soil / Cd / 0.07 / 0.11 / - / - / 0.10 / - / -
Fe / 5729 / 504 / 354 / 7873 / 2.6 / 124 / 5685
Mn / 43 / 40 / 121 / 1698 / 5.6 / 37 / 697
Zn / 31 / 4 / b.d. / b.d. / 3.9 / 20 / 9
Table S4:Gene expression data for different rice genotypes grown in unflooded (A) and continuously flooded (C) potted soil conditions.Expression of OsIRT1 and IDEF1 was measured in the youngest expanded leaf (at tillering) or in selected flag-leaves (at flowering stage). Gene expression is expressed relative to a combined sample cDNA pool, and is normalised against expression of eEF1α. Mean values are shown (n=4) with associated SE. All differences between treatments were not statistically significant.
OsIRT1 / IDEF1
Tillering / Early Flwr / Late Flwr / Tillering / Late Flwr
Mean / SE / Mean / SE / Mean / SE / Mean / SE / Mean / SE
HBK A / 0.91 / 0.14 / 1.29 / 0.11 / 1.83 / 0.24
HBK C / 0.73 / 0.25 / 1.45 / 0.07 / 1.48 / 0.09
BDN A / 1.13 / 0.24 / 0.78 / 0.26 / 1.96 / 0.45
BDN C / 0.94 / 0.11 / 0.65 / 0.06 / 1.62 / 0.30
MLY A / 1.39 / 0.46 / 1.88 / 0.11
MLY C / 0.94 / 0.19 / 1.95 / 0.50
KAS A / 0.50 / 0.11 / 0.43 / 0.05 / 1.49 / 0.04
KAS C / 0.52 / 0.09 / 0.35 / 0.07 / 2.03 / 0.15
KSH A / 1.16 / 0.22
KSH C / 1.26 / 0.09
NPB A / 1.38 / 0.19 / 0.94 / 0.02 / 10.05 / 4.78
NPB C / 1.57 / 0.08 / 1.48 / 0.09 / 10.57 / 6.84
Figure S1: Expression analysis performed in rice roots during the initial selection of these transgenic plants, compared with WT. Expression in (a) OsNRAMP1-RNAi plants, as well as (b) OsNRAMP1-OX plants, relative to α-tubulin expression. Data was provided by the Laboratory of Plant Biotechnology at the Graduate School of Agricultural and Life Sciences, the University of Tokyo, Japan.
References
Arao T, Ae N (2003) Genotypic variations in cadmium levels of rice grain. Soil Sci Plant Nutr 49:473-479
Ishikawa S, Abe T, Kuramata M, Yamaguchi M, Ando T, Yamamoto T, Yano M (2010) 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 61:923-934. doi:10.1093/jxb/erp360
Ishikawa S, Ae N, Yano M (2005) Chromosomal regions with quantitative trait loci controlling cadmium concentration in brown rice (Oryza sativa). New Phytol 168:345-350
Kashiwagi T, Shindoh K, Hirotsu N, Ishimaru K (2009) Evidence for separate translocation pathways in determining cadmium accumulation in grain and aerial plant parts in rice. BMC Plant Biol 9:8
Kobayashi T, Itai RN, Ogo Y, Kakei Y, Nakanishi H, Takahashi M, Nishizawa NK (2009) The rice transcription factor IDEF1 is essential for the early response to iron deficiency, and induces vegetative expression of late embryogenesis abundant genes. Plant J 60:948-961
Murakami M, Ae N, Ishikawa S, Ibaraki T, Ito M (2008) Phytoextraction by a high-Cd-accumulating rice: reduction of Cd content of soybean seeds. Environ Sci Technol 42:6167-6172
Ueno D, Kono I, Yokosho K, Ando T, Yano M, Ma JF (2009a) A major quantitative trait locus controlling cadmium translocation in rice (Oryza sativa). New Phytol 182
Ueno D, Koyama E, Kono I, Ando T, Yano M, Ma JF (2009b) Identification of a novel major quantitative trait locus controlling distribution of Cd between roots and shoots in rice. Plant Cell Physiol 50:2223-2233. doi:10.1093/pcp/pcp160
Uraguchi S, Mori S, Kuramata M, Kawasaki A, Arao T, Ishikawa S (2009) Root-to-shoot Cd translocation via the xylem is the major process determining shoot and grain cadmium accumulation in rice. J Exp Bot 60:2677-2688. doi:10.1093/jxb/erp119
Yan Y-F, Choi D-H, Kim D-S, Lee B-W (2010) Absorption, translocation, and remobilization of cadmium supplied at different growth stages of rice. J Crop Sci Biotech 13:113-119. doi:10.1007/s12892-010-0045-4