NIP aquaporins: new tools to reduce rice arsenic accumulation

Lead Research Organisation: Rothamsted Research
Department Name: Sustainable Soils and Grassland Systems

Abstract

Rice is the staple food for about half of the world's population. Among major food crops, rice is especially efficient at the accumulation of arsenic which is toxic and carcinogenic. This accumulation presents a potentially serious health risk, because consumption of rice contributes a large proportion of inorganic As intake for people living on a rice-diet anywhere in the world. The problem is exacerbated in many rice-producing regions by the past use of arsenic-based herbicides and insecticides, mining, and irrigation with arsenic-contaminated groundwater. There is an urgent need to develop strategies to reduce this widespread contamination of the food chain. This requires a better understanding of the mechanisms responsible for uptake, transport and distribution of arsenic into rice grain. Unlike aerobic soils where arsenate is the predominant chemical species of arsenic, the arsenite form dominates in the reducing environment of flooded paddy soils. We have recently discovered that arsenite is taken up by rice roots through the silicon uptake pathway. Rice accumulates a large amount of silicon, which protects the plant against biotic and abiotic stresses. An aquaporin channel protein called NIP2;1 transports silicon, and also inadvertently arsenite, into the root cells. There is a family of 10 NIP proteins in rice, some of which are expressed mainly in leaf and grain tissues. We hypothesise that some of these NIP channel proteins are involved in arsenic transport to the rice grain. We will evaluate the role of NIP proteins in arsenic distribution to the leaf and rice grain using a range of molecular and physiological methods. We will investigate the pattern of expression of different NIP genes in leaf and grain tissues during grain development and the transport function of NIP proteins for arsenic compounds. We will determine the specificity of different NIP proteins for arsenic compounds and manipulate the amino acid composition in a key region of the proteins to alter their transport selectivity for arsenic. Results obtained from this project will provide insight into the mechanisms of arsenic transport in plants and help the development of counter measures to reduce arsenic accumulation in rice grain through molecular breeding or transgenic approaches.

Technical Summary

Paddy rice accumulates more arsenic than other cereal crops because arsenite is mobilised in flooded soil and then taken up by the highly efficient silicon uptake pathway. We have recently discovered that the aquaporin NIP2;1 (Nodulin 26-like Intrinsic Protein), a silicon transporter, also mediates the influx of arsenite into root cells of rice. Preliminary evidence suggests that arsenite permeability is more widespread among NIP proteins than silicon permeability. Based on preliminary evidence that we have obtained, we hypothesise that NIP proteins are involved in the distribution of arsenite and methylated arsenic species to leaf and grain tissues and that the relative specificity for arsenic and silicon is determined mainly by the amino acid composition of the aromatic/arginine filter of the NIP proteins. We will investigate the functions and substrate specificity of different rice NIP proteins using heterologous systems of Xenopus oocytes and yeast, and establish the structure-function relationship using site-directed mutagenesis to manipulate the amino acid composition of the aromatic/arginine filter. We will evaluate the in planta functions of rice NIP genes in terms of arsenic distribution in leaf and grain tissues using loss of function mutants and transgenic plants with altered expression of specific genes. This proposal will provide important insight into the mechanism of arsenic transport in plants, particularly toward rice grain. The results can be used to develop counter measures to reduce arsenic accumulation in rice grain through molecular breeding or transgenic approaches.

Planned Impact

Who will benefit from this research? Rice is a staple food for about half of the world population. Excessive accumulation of arsenic in rice poses a significant problem in terms of food chain contamination which has adverse impacts on human health. Recent studies have shown that intake of arsenic from rice can significantly elevate cancer risk. Methods to reduce arsenic accumulation will benefit people in the general public anywhere in the world who consume rice. This includes babies and children who consume rice-based products; in fact they are the most vulnerable group to arsenic contamination. Reduced arsenic accumulation by rice is likely to enhance the crop tolerance to arsenic and increase yields under arsenic stress; this can bring benefit to rice farmers in parts of Asia and Africa where soil and irrigation water are contaminated with arsenic, leading to a more sustainable use of natural resources. How will they benefit from this research? The proposed research will address the fundamental question of the mechanism of transport and distribution of arsenic to rice grain, which is currently poorly understood. This knowledge is essential for devising strategies to develop low arsenic rice. It is envisaged that key NIP genes involved in arsenic distribution to rice grain will be identified, and the structure-function relationship governing the selectivity of arsenic by NIP proteins established in this project. This information will greatly aid the development of low arsenic rice through molecular breeding or transgenic approaches. The impact of low arsenic rice on human health will be substantial and long-term. Use of GM technology to produce rice plants that avoid the accumulation of arsenic in the grain will be very important for improving public opinion on the beneficial use of this technology in food production and provision of a healthier diet. There will be a clear and simple advantage to health resulting from the plants produced in this research, potentially benefitting some of the poorer parts of the world. This research has the potential to grab the public's imagination and can be simply presented by the media. What will be done to ensure that they benefit from this research? Potential intellectual property will be identified and properly protected; Rothamsted and the University of York have offices to advise on this process. Non-patentable findings will be disseminated to the scientific community through conference presentations and peer-reviewed publications; to agronomists and crop breeders through a workshop, and our existing links with the south and southeast Asian countries and the International Rice Research Institute; and the wider public through the media, national and local events. Material Transfer Agreements will be arranged between the partners in this proposal. MTAs represent the first step in a process to safeguard the ownership and rights of the research organisations involved, liabilities and to cover any IP arising. This also covers other important aspects of working together such as acknowledgement of the source of the materials in any publication, provision of raw data, reports or publications or inventions relating to the materials and arising from the specific research programme. The agreement also requires subsequent discussion of any intellectual property arising, and negotiation on how it should be exploited, with what division of work related to its exploitation and the revenues between the partners. The supplier of materials will retain the right to use an Invention for non-commercial research purposes. The PI and Co/Is have relevant experience and track record in public dissemination of research findings. Any publicity activities will be coordinated through the Science Communication offices in both host organisations. Examples include recent highlights and interviews on our arsenic research in Nature, Science, New York Times, other websites, TV and radio.

Publications


10 25 50
 
Description 1. We have systematically investigated the expression pattern, tissue localisation, permeability to arsenite and silicon, and in planta functions of a number of rice NIP genes. NIP2;1 was highly expressed in rice roots and the expression was strongly down-regulated by arsenite exposure. NIP1;1 and NIP3;2 were expressed at moderate levels in rice roots, and the expression of NIP3;2 was strongly up-regulated by exposure to high concentrations of arsenite. NIP1;1 and NIP3;3 were expressed at high levels in developing grain. All NIPs tested showed permeability to arsenite when expressed in Xenopus laevis oocytes. In contrast, permeability to silicon was restricted to NIP2;1 and NIP3;3 only. NIP2;1, NIP1;1 and NIP3;3 were also permeable to methylated arsenic MMA. We further demonstrated that the aromatic/arginine filter in the aquaporin proteins is crucial for silicon/arsenite selectivity. Changing a single amino acid residue in the NIP3;2 filter (AAAR) to that of the NIP3;3 filter (AIAR) made NIP3;2 permeable to silicon. This new knowledge can be further explored to maximise silicon transport while suppressing arsenite accumulation. Using knockout mutants, we showed that NIP3;2, which was strongly expressed in the lateral roots, made a small but significant contribution to the uptake of arsenite by rice roots, whilst NIP3;3 played a significant role in the accumulation of arsenite in rice grain. This new knowledge extends our previous finding that NIP2;1 plays an important role in arsenite uptake by rice roots.
2. During the course of this project, we have established a strong collaboration with Chris Grosvenor and Katie Moore of the Materials Department, Oxford University, and Tina Geraki and Fred Mosselmans of the Diamond Light Source. Through this collaboration, we established methods for imaging arsenic and other trace elements in plant tissues at the cellular and sub-cellular levels using synchrotron X-ray fluorescence and high resolution secondary mass spectrometry. We demonstrated that arsenic is sequestered as As-thiol complexes in the vacuoles of the pericycle and endodermal cells in the root, and the phloem companion cells in the vascular bundles in the stems, nodes and leaves of rice. Our results showed that rice nodes plays a crucial role in filtering arsenite restricting its movement to rice grain, and the silicon/arsenite efflux transporter Lsi2 is involved in the distribution of arsenite towards grain. This new knowledge suggests that enhancing the synthesis of phytochelatins in rice roots and nodes can decrease the distribution of arsenic to rice grain.
3. We have previously shown that following the uptake of arsenate via phosphate transporters, it is rapidly reduced to arsenite with the majority of arsenite being extruded to the external medium. This arsenite efflux prevents excessive build-up of arsenic in plant tissues and is an important mechanism of arsenic detoxification. We found that NIP2;1 (Lsi1) is a bidirectional transporter for arsenite and can mediate both the uptake and efflux of arsenite from rice roots. In collaboration with David Salt of Aberdeen University, we have characterised the function of a new arsenate enzyme, HAC1, in Arabidopsis thaliana. This enzyme is localised to the epidermal cells of roots and functions as an arsenate reductase to allow arsenite to be extruded into the soil. Loss of function of HAC1 leads to abolition of arsenite efflux and arsenic hyperaccumulation in the shoots. HAC1 therefore plays a key role in limiting arsenic accumulation in plants. This discovery opens a new way to block arsenic accumulation in the edible parts of crops.
Exploitation Route The European Union will introduce very strict limits of arsenic in rice from next year, 0.1 ppm for rice used in baby food and 0.2 ppm for other consumers. A large proportion of rice produced worldwide is likely to exceed these limits because the global mean of As concentration in rice is 0.15 ppm. Based on the findings of this and our other projects on arsenic, we have proposed a number of solutions to decrease arsenic accumulation in rice, including use of silicon fertilisers to suppress arsenic translocation to the grain. Some of these solutions can be adopted by rice growers (e.g. silicon fertilisers and water management), whereas others (e.g. breeding and genetic engineering) require further research and development from academic and rice breeders.
Sectors Agriculture, Food and Drink,Environment
 
Description The European Union will introduce very strict limits of arsenic in rice from next year, 0.1 ppm for rice used in baby food and 0.2 ppm for other consumers. A large proportion of rice produced worldwide is likely to exceed these limits because the global mean of As concentration in rice is 0.15 ppm. Our research findings and related publications have been extensively referred to in a recent Discussion paper on possibility to develop a code of practice for the prevention and reduction of arsenic contamination in rice by the Joint FAO/WHO Food Standards Programme (2013).
First Year Of Impact 2013
Sector Agriculture, Food and Drink
Impact Types Policy & public services
 
Description Arsenic as a Food Chain contaminant: mechanisms of plant uotake and metabolism and mitigation strategies. 
Organisation University of Aberdeen
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution A range of mitigation methods, from agronomic measures and plant breeding to genetic modification, may be employed to reduce As uptake by food crops.
Start Year 2009
 
Description Arsenic as a Food Chain contaminant: mechanisms of plant uptake and metabolism and mitigation stratergies. 
Organisation University of Aberdeen
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution no description available
Start Year 2009
 
Description Heterologous expression of the yeast arsenite efflux system ACR3 improves Arabidopsis thaliana to arsenic stress. 
Organisation University of York
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution This approach would enhance the growth potential of crops in environments in which As is present at toxic levels, but further fine tuning may be required, for example in the form of tissue-specific promoters, to avoid potentially harmful effects, such as greater root to shoot translocation of As. The latter may lead to increased As accumulation in grain, which is undesirable for food crops. However, increased root to shoot translocation provides a means to increase shoot As content, which could greatly benefit phytoremediation applications.
Start Year 2010
 
Description Heterologous expression of the yeast arsenite efflux system ACR3 improves Arabidopsis thaliana tolerance to arsenic stress 
Organisation Institute of Food Biotechnology and Genomics, Osipovs Logo
Country Ukraine 
Sector Public 
PI Contribution This approach would enhance the growth potential of crops in environments in which As is present at toxic levels, but further fine tuning may be required, for example in the form of tissue-specific promoters, to avoid potentially harmful effects, such as greater root to shoot translocation of As. The latter may lead to increased As accumulation in grain, which is undesirable for food crops. However, increased root to shoot translocation provides a means to increase shoot As content, which could greatly benefit phytoremediation applications.
Start Year 2010
 
Description Heterologous expression of the yeast arsenite efflux system ACR3 improves Arabidopsis thaliana tolerance to arsenic stress 
Organisation Institute of Food Biotechnology and Genomics, Osipovs Logo
Country Ukraine 
Sector Public 
PI Contribution This approach would enhance the growth potential of crops in environments in which As is present at toxic levels, but further fine tuning may be required, for example in the form of tissue-specific promoters, to avoid potentially harmful effects, such as greater root to shoot translocation of As. The latter may lead to increased As accumulation in grain, which is undesirable for food crops. However, increased root to shoot translocation provides a means to increase shoot As content, which could greatly benefit phytoremediation applications
Start Year 2010
 
Description Heterologous expression of the yeast arsenite efflux system ACR3 improves Arabidopsis thaliana tolerance to arsenic stress. 
Organisation University of York
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution This approach would enhance the growth potential of crops in environments in which As is present at toxic levels, but further fine tuning may be required, for example in the form of tissue-specific promoters, to avoid potentially harmful effects, such as greater root to shoot translocation of As. The latter may lead to increased As accumulation in grain, which is undesirable for food crops. However, increased root to shoot translocation provides a means to increase shoot As content, which could greatly benefit phytoremediation applications
Start Year 2010
 
Description High-Resolution Secondary Ion Mass Spectrometry Reveals the Contrasting Subcellular Distribution of arsenic and silicon in rice roots. 
Organisation Oxford Brookes University
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution This study reveals the vacuolar sequestration of As in rice roots and contrasting patterns of As and Si subcellular localization, despite both being transported across the plasma membranes by the same transporters
Start Year 2010
 
Description High-Resolution Secondary Ion Mass Spectrometry Reveals the Contrasting Subcellular Distribution of arsenic and silicon in rice roots. 
Organisation University of Oxford
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution This study reveals the vacuolar sequestration of As in rice roots and contrasting patterns of As and Si subcellular localization, despite both being transported across the plasma membranes by the same transporters.
Start Year 2010
 
Description High-Resolution Secondary Ion Mass Spectrometry reveals the contrasting subcellular distribution of arsenic and silicon in rice roots. 
Organisation Oxford Brookes University
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution This study reveals the vacuolar sequestration of As in rice roots and contrasting patterns of As and Si subcellular localization, despite both being transported across the plasma membranes by the same transporters.
Start Year 2010
 
Description High-Resolution Secondary Ion Mass Spectrometry reveals the contrasting subcellular distribution of arsenic and silicon in rice roots. 
Organisation University of Oxford
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution This study reveals the vacuolar sequestration of As in rice roots and contrasting patterns of As and Si subcellular localization, despite both being transported across the plasma membranes by the same transporters.
Start Year 2010
 
Description Knocking Out ACR2 Does Not Affect Arsenic Redox Status in Arabidopsis thaliana: Implications 
Organisation University of Aberdeen
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Our results suggest the existence of multiple pathways of arsenate reduction in plants and yeast.
Start Year 2010
 
Description Knocking Out ACR2 Does Not Affect Arsenic Redox Status in Arabidopsis thaliana: Implications 
Organisation University of Saskatchewan
Country Canada 
Sector Academic/University 
PI Contribution Our results suggest the existence of multiple pathways of arsenate reduction in plants and yeast.
Start Year 2010
 
Description Knocking Out ACR2 Does Not Affect Arsenic Redox Status in Arabidopsis thaliana: Implications for As Detoxification and Accumulation in Plants 
Organisation Free University of Amsterdam
Country Netherlands, Kingdom of the 
Sector Academic/University 
PI Contribution Our results suggest the existence of multiple pathways of arsenate reduction in plants and yeast.
Start Year 2010
 
Description Knocking Out ACR2 Does Not Affect Arsenic Redox Status in Arabidopsis thaliana: Implications for AsDetoxification and Accumulation in Plants 
Organisation Agricultural University of Hebei
Country China, People's Republic of 
Sector Academic/University 
PI Contribution Our results suggest the existence of multiple pathways of arsenate reduction in plants and yeast.
Start Year 2010
 
Description The aromatic/arginine selectivity filter of NIP aquaporins plays a critical role in substrate selectivity for silicon boron, and arsenic 
Organisation Okayama University
Country Japan 
Sector Academic/University 
PI Contribution The results reveal that the residue at the H5 position of the ar/R filter of both OsLsi1 and AtNIP5;1 plays a key role in the permeability to Si and B, but there is a relatively low selectivity for arsenite.
Start Year 2010
 
Description The aromatic/arginine selectivity filter of NIP aquaporins plays a critical role in substrate selectivity for silicon, boron, and arsenic 
Organisation Okayama University
Country Japan 
Sector Academic/University 
PI Contribution The results reveal that the residue at the H5 position of the ar/R filter of both OsLsi1 and AtNIP5;1 plays a key role in the permeability to Si and B, but there is a relatively low selectivity for arsenite.
Start Year 2010
 
Description Function of NIP aquaporin proteins in arsenic accumulation in rice 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk given at 16th International workshop on Plant Membrane Biology, Kurashiki, Japan, 26-31 March 2013, Title:

no actual impacts realised to date
Year(s) Of Engagement Activity 2013
 
Description Investigating Arsenic Transport in Rice 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk given at Plant Transport Group Meeting, Lancaster, 14-16 September 2011

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Investigating Arsenic Transport in Rice 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Talk given at Plant Transport Group Meeting, Lancaster, 14-16 September 2011

no actual impacts realised to date
Year(s) Of Engagement Activity 2011