Please wait a minute...
Journal of Integrative Agriculture  2019, Vol. 18 Issue (3): 688-697    DOI: 10.1016/S2095-3119(18)61904-5
Agro-ecosystem & Environment Advanced Online Publication | Current Issue | Archive | Adv Search |
Reduction in cadmium accumulation in japonica rice grains by CRISPR/Cas9-mediated editing of OsNRAMP5
YANG Chang-hong1, 2*, ZHANG Yang1, 2*, HUANG Chao-feng1, 2
1 College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, P.R.China
2 Shanghai Center for Plant Stress Biology and National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, P.R.China
Download:  PDF (625KB) ( )  
Export:  BibTeX | EndNote (RIS)      
Cadmium (Cd) intake is harmful to human health and Cd contamination in rice grains represents a severe threat to those consuming rice as a staple food.  Knockout of Cd transporters is a promising strategy to reduce Cd accumulation in rice grains.  OsNRAMP5 is the major transporter for Cd and manganese (Mn) uptake in rice.  Nevertheless, it is uncertain whether knockout of OsNRAMP5 is applicable to produce low Cd rice without affecting plant growth and grain yield.  In this study, we adopted CRISPR/Cas9-based gene editing technology to knock out OsNRAMP5 in two japonica varieties.  We generated three independent transgene-free osnramp5 mutants and investigated the effect of osnramp5 mutations on Cd accumulation and plant growth.  Hydroponic experiments showed that plant growth and chlorophyll content were significantly reduced in osnramp5 mutants at low Mn conditions, and this defective growth in the mutants could be fully rescued by supply of high levels of Mn.  Cd and Mn accumulation in both roots and shoots was markedly reduced in the mutants compared to that in wild-type plants.  In paddy field experiments, although Cd in flag leaves and grains was greatly reduced in osnramp5 mutants, some agronomic traits including plant height, seed setting rate, and grain number per panicle were affected in the mutants, which ultimately caused a mild reduction in grain yield.  The reduced plant growth in the mutants can be attributed to a marked decrease in Mn accumulation.  Our results reveal that the manipulation of OsNRAMP5 should be treated with caution: When assessing the applicability of osnramp5 mutants, soil pH and soil water content in paddy fields need to be taken into consideration, since they might affect the levels of available Mn in the soil and consequently determine the effect of the mutation on grain yield.
Keywords:  cadmium content        CRISPR/Cas9        OsNRAMP5        Oryza sativa (rice)  
Received: 05 January 2018   Accepted:
Fund: This work was supported by the Key Technologies R&D Program of China during the 12th Five-year Plan period (2015BAD05B04), the Jiangsu Science Fund for Distinguished Young Scholars, China (BK20150027), the Strategic Priority Research Program of Chinese Academy of Sciences (XDPB0404) and the Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences.
Corresponding Authors:  Correspondence HUANG Chao-feng, Tel: +86-21-54924320, E-mail:   
About author:  YANG Chang-hong, E-mail:; ZHANG Yang, E-mail:; * These authors contributed equally to this study.

Cite this article: 

YANG Chang-hong, ZHANG Yang, HUANG Chao-feng. 2019. Reduction in cadmium accumulation in japonica rice grains by CRISPR/Cas9-mediated editing of OsNRAMP5. Journal of Integrative Agriculture, 18(3): 688-697.

Bortesi L, Fischer R. 2015. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnology Advances, 33, 41–52.
Clemens S, Aarts M G M, Thomine S, Verbruggen N. 2013. Plant science: The key to preventing slow cadmium poisoning. Trends in Plant Science, 18, 92–99.
Clemens S, Palmgren M G, Kramer U. 2002. A long way ahead: Understanding and engineering plant metal accumulation. Trends in Plant Science, 7, 309–315.
Fujimaki S, Suzui N, Ishioka N S, Kawachi N, Ito S, Chino M, Nakamura S. 2010. Tracing cadmium from culture to spikelet: noninvasive imaging and quantitative characterization of absorption, transport, and accumulation of cadmium in an intact rice plant. Plant Physiology, 152, 1796–1806.
Ishikawa S, Ishimaru Y, Igura M, Kuramata M, Abe T, Senoura T, Hase Y, Arao T, Nishizawa N K, Nakanishi H. 2012. Ion-beam irradiation, gene identification, and marker-assisted breeding in the development of low-cadmium rice. Proceedings of the National Academy of Sciences of the United States of America, 109, 19166–19171.
Jarup L, Akesson A. 2009. Current status of cadmium as an environmental health problem. Toxicology and Applied Pharmacology, 238, 201–208.
Li M R, Li X X, Zhou Z J, Wu P Z, Fang M C, Pan X P, Lin Q P, Luo W B, Wu G J, Li H Q. 2016. Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in rice using a CRISPR/Cas9 system. Frontiers in Plant Science, 7, 377.
Ma X L, Zhang Q Y, Zhu Q L, Liu W, Chen Y, Qiu R, Wang B, Yang Z F, Li H Y, Lin Y R, Xie Y Y, Shen R X, Chen S F, Wang Z, Chen Y L, Guo J X, Chen L T, Zhao X C, Dong Z C, Liu Y G. 2015. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Molecular Plant, 8, 1274–1284.
Nawrot T S, Staessen J A, Roels H A, Munters E, Cuypers A, Richart T, Ruttens A, Smeets K, Clijsters H, Vangronsveld J. 2010. Cadmium exposure in the population: From health risks to strategies of prevention. Biometals, 23, 769–782.
Nevo Y, Nelson N. 2006. The NRAMP family of metal-ion transporters. Biochimica et Biophysica Acta - Molecular Cell Research, 1763, 609–620.
Satarug S, Garrett S H, Sens M A, Sens D A. 2010. Cadmium, environmental exposure, and health outcomes. Environmental Health Perspectives, 118, 182–190.
Sasaki A, Yamaji N, Yokosho K, Ma J F. 2012. Nramp5 is a major transporter responsible for manganese and cadmium uptake in rice. The Plant Cell, 24, 2155–2167.
Satoh-Nagasawa N, Mori M, Nakazawa N, Kawamoto T, Nagato Y, Sakurai K, Takahashi H, Watanabe A, Akagi H. 2012. Mutations in rice (Oryza sativa) heavy metal ATPase 2 (OsHMA2) restrict the translocation of zinc and cadmium. Plant and Cell Physiology, 53, 213–224.
Schmidt S B, Jensen P E, Husted S. 2016. Manganese deficiency in plants: The impact on photosystem II. Trends in Plant Science, 21, 622–632.
Sun Y W, Zhang X, Wu C Y, He Y B, Ma Y Z, Hou H, Guo X P, Du W M, Zhao Y D, Xia L Q. 2016. Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate synthase. Molecular Plant, 9, 628–631.
Takahashi R, Ishimaru Y, Shimo H, Ogo Y, Senoura T, Nishizawa N K, Nakanishi H. 2012. The OsHMA2 transporter is involved in root-to-shoot translocation of Zn and Cd in rice. Plant Cell and Environment, 35, 1948–1957.
Tang L, Mao B G, Li Y K, Lv Q M, Zhang L P, Chen C Y, He H J, Wang W P, Zeng X F, Shao Y, Pan Y L, Hu Y Y, Peng Y, Fu X Q, Li H Q, Xia S T, Zhao B R. 2017. Knockout of OsNramp5 using the CRISPR/Cas9 system produces low Cd-accumulating indica rice without compromising yield. Scientific Reports, 7, 14438.
Tsukahara T, Ezaki T, Moriguchi J, Furuki K, Shimbo S, Matsuda-Inoguchi N, Ikeda M. 2003. Rice as the most influential source of cadmium intake among general Japanese population. Science of the Total Environment, 305, 41–51.
Ueno D, Yamaji N, Kono I, Huang C F, Ando T, Yano M, Ma J F. 2010. Gene limiting cadmium accumulation in rice. Proceedings of the National Academy of Sciences of the United States of America, 107, 16500–16505.
Uraguchi S, Kamiya T, Sakamoto T, Kasai K, Sato Y, Nagamura Y, Yoshida A, Kyozuka J, Ishikawa S, Fujiwara T. 2011. Low-affinity cation transporter (OsLCT1) regulates cadmium transport into rice grains. Proceedings of the National Academy of Sciences of the United States of America, 108, 20959–20964.
Watanabe T, Zhang Z W, Moon C S, Shimbo S, Nakatsuka H, Matsuda-Inoguchi N, Higashikawa K, Ikeda M. 2000. Cadmium exposure of women in general populations in Japan during 1991–1997 compared with 1977–1981. International Archives of Occupational and Environmental Health, 73, 26–34.
Yang M, Zhang Y Y, Zhang L J, Hu J T, Zhang X, Lu K, Dong H X, Wang D J, Zhao F J, Huang C F, Lian X M. 2014. OsNRAMP5 contributes to manganese translocation and distribution in rice shoots. Journal of Experimental Botany, 65, 4849–4861.
Zhang Y L, Ma X L, Xie X R, Liu Y G. 2017. CRISPR/Cas9-based genome editing in plants. Gene Editing in Plants, 149, 133–150.
Zhao F J, Ma Y B, Zhu Y G, Tang Z, McGrath S P. 2015. Soil contamination in China: Current status and mitigation strategies. Environmental Science & Technology, 49, 750–759.
[1] JIN Ming-hui, TAO Jia-hui, LI Qi, CHENG Ying, SUN Xiao-xu, WU Kong-ming, XIAO Yu-tao . Genome editing of the SfABCC2 gene confers resistance to Cry1F toxin from Bacillus thuringiensis in Spodoptera frugiperda[J]. >Journal of Integrative Agriculture, 2021, 20(3): 815-820.
[2] ZHANG Ting-ting, WEN Ting-mei, YUE Yang, YAN Qiang, DU Er-xia, FAN San-hong, Siegfried ROTH, LI Sheng, ZHANG Jian-zhen, ZHANG Xue-yao, ZHANG Min. Egg tanning improves the efficiency of CRISPR/Cas9-mediated mutant locust production by enhancing defense ability after microinjection[J]. >Journal of Integrative Agriculture, 2021, 20(10): 2716-2726.
[3] ZHANG Ru, ZHANG Zhong-jie, YU Ye, HUANG Yong-ping, QIAN Ai-rong, TAN An-jiang. Proboscipedia and Sex combs reduced are essential for embryonic labial palpus specification in Bombyx mori[J]. >Journal of Integrative Agriculture, 2020, 19(6): 1482-1491.
[4] LIU Hui, LIU Chang, ZHAO Yu-hang, HAN Xue-jie, ZHOU Zheng-wei, WANG Chen, LI Rong-feng, LI Xue-ling . Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts[J]. >Journal of Integrative Agriculture, 2018, 17(2): 406-414.
No Suggested Reading articles found!