Final Report Summary - ORYZAQUA (Cell biology of rice aquaporins)
Introduction
Global changes are predicted to dramatically increase the variability of water supply, both in space and time, and will, thus affect crop yield. Several Mediterranean regions are already under severe risk of drought, extreme temperatures, and of other types of abiotic stress linked to water (e.g. as a result of periodic flooding, or salt stress). Southern countries such as Vietnam have also to face global changes. For instance, compared to the period 1980-2000, climate models predict an increase in temperature in the range of 1.2°C– 4.5°C in the North and in the range of 0.5°C – 3.0°C in the South and a sea level raise as high as 15 – 90 cm along the coastline of Vietnam, for 2070. Since Vietnam rice production is mainly located in the two deltas of Red river and Mekong, this sea level raise will be detrimental. Furthermore, in the Vietnam mountainous regions, the rice production relies essentially on rain fed watering. Changing the regime of watering is also detrimental. Plants are subjected to numerous abiotic stresses such as drought, salinity, extreme temperatures s, nutrient deficiencies and mineral toxicities, reducing their growth and therefore having a major impact on crop yield. Unfortunately, the adaptive mechanisms which allow some plants to cope with abiotic stresses are poorly understood. The control of water flow from the soil to the atmosphere via the root-to-stomata continuum is a central mechanism for plant growth and survival. More specifically, water transport in plant roots has been the object of intense research1. It was shown that water channel proteins named aquaporins mediate a significant part of the root hydraulic conductivity (Lpr) in numerous plant species including the model plant Arabidopsis thaliana and crops such as rice, maize, grapevine, or legumes2. Several of the above mentioned stresses are known to impact the water transport properties of plant tissues and to act on aquaporin regulation2.
ORYZAQUA project is willing to focus on rice (Oryza sativa) for understanding the function and regulation of aquaporins at the cellular level, using root cells of rice plantlets as a model to address the effects of salinity stress. Here, we want to focus on plantlets two weeks after germination, since the transplanting in the paddy fields is proceeded at this critical developmental stage when the plants are potentially subjected to severe stresses. The first aim is to address the water transport function of rice aquaporins in the root system. Next, we will monitor their subcellular localization by means of cell biology approaches.
Main results
Water transport function of aquaporins in the rice root system
We used a pressure-chamber system to monitor root hydraulic conductivity (Lpr)3. We compared two genotypes with contrasted watering regimes: Nipponbare is an irrigated lowland japonica rice and its genome sequence is now available; Azucena is a rainfed upland rice variety. Rice compared to Arabidopsis, presents a root architecture characterized by a fibrous system comprising four types of roots (the radicle, the crown roots, the large lateral roots, and the small lateral roots). These different types of roots are morphologically and anatomically distinct. To take into account the contribution of crown-root type, the genotype crl1, standing for crown-root-less mutant, was also analysed. Contrasted Lpr values were observed between genotypes (Figure 1). The two lowland and upland genotypes showed a marked difference. Interestingly, the mutant crl1 showed the lowest Lpr value. The salt-stress effect on the Lpr was also monitored. We observed a decrease of these values, indicating that salt has strong and fast inhibiting effects on the water transport capacities of rice plantlets.
Subcellular localization of rice aquaporins and sub-cellular markers
Here, we monitored the sub-cellular localization of selected rice aquaporins, the Plasma membrane Intrinsic Proteins (OsPIPs), by means of confocal laser scanning microscopy. For this, we cloned the cDNA sequences of four most highly root-expressed isoforms OsPIP1;1, OsPIP2;1, OsPIP2;4, and OsPIP2;54 and fused them individually with the sequence of the green fluorescent protein (GFP), under the control of a Cauliflower Mosaic Virus 35S promoter. The constructs were carried by a binary vector and were tested by the agroinfiltration method which allows a rapid observation of the localization of the transgenic products5. The constructs labelled the periphery of the cells indicating a plasma membrane (PM) localization, consistent with the nomenclature of these isoforms (Figure 2A-2E). OsRab5, a marker of the pre-vacuolar compartments (PVC)6 was expressed as a protein fusion with the fluorescent protein mCherry and showed a labelling surrounding some intracellular compartments such as small vacuoles (Figure 2F). We were also curious of the sub-cellular localization of the construct in Arabidopsis thaliana. For this, the floral-dip method was employed for obtaining Arabidopsis transgenic plants stably transformed and expressing individual OsPIP-GFP constructs (Figure 3). These constructs showed a peripheral labelling in agreement with the expected localization in the PM. Interestingly, under salt stress, a strong intracellular labelling was observed (Figure 3), reminiscent of what was observed for Arabidopsis PIP constructs in a previous study7. OsRab5-construct expression in Arabidopsis labelled intracellular compartments in control and salt-stressed plants (Figure 3). Finally, the Nipponbare genotype was stably transformed by Agrobacterium via a calli co-cultivation method8. Transgenic plants expressing OsPIP1;1-GFP or OsPIP2;1-GFP were obtained and the sub-cellular localization of OsPIP1;1-GFP was shown here as representative of the family members. In control conditions (Figure 4 upper panels), we observed a strict labelling of the PM for all the cells amenable to confocal microscopy analysis. Interestingly, the same pattern of localization was observed after a salt-stress (Figure 4 lower panels). We concluded that there is no detectable relocalization of OsPIP1;1-GFP construct upon 100 mM NaCl treatment.
Conclusion
This work establishes for the first time the strong inhibitory effect of salinity on the root water transport of rice plantlets. It is interpreted as a physiological mechanism to fastly (< 1 hour) respond to environmental challenges. Also, since this inhibitory effect is fast, it points to the regulation of aquaporin activity as a major factor of the whole-plant response to salinity. Interestingly, under salinity, we did not observe any differential localization of OsPIP1;1, taken here as a representative of PIP aquaporin isoforms. This would suggest a mechanism independent of PM aquaporin internalization to account for the reduction in Lpr.
Potential impact and use and any socio-economic impact
In the next 20 years, it is predicted that nearly half of the world will be living under severe water stress. We believe that reducing water needs through plant science technologies is feasible. Besides improved water management techniques, crops with improved water use efficiency may be developed. For example, a U.S. study found that fewer water is needed to grow irrigated cotton, corn, soybean and wheat today, compared to 20 years ago 9. Furthermore, developing drought tolerant crops is of importance and feasible (see this example of a corn tolerant to drought which is being developed for Africa: http://www.aatf-africa.org/wema/en/). ORYZAQUA will help enhance scientific knowledge on topics of crucial socio-economic importance. The agricultural production indeed needs to be better adapted to erratic, unpredictable environmental conditions that will arise under climate changes. This will allow the development of robust crops with improved traits for biomass yield, productivity and quality under adverse environmental conditions. Therefore, the issues that the proposal is intended to address are (1) increasing demand for plants for food uses and (2) interaction between climate change and crops.
ORYZAQUA will help establish major outputs and outcomes in several ways:
1. It addresses water stress affecting crop plants and will develop agricultural plant varieties better equipped to withstand water stress, useful for production of biomass and bioproducts.
2. It targets commercially important crops using state-of-the-art knowledge on physiological, molecular and genetic processes obtained on the model plant Arabidopsis. This knowledge, which relates to plant tolerance and adaptation to water stress, will be used for ultimately developing robust crops with improved traits for biomass yield, productivity and quality under adverse and/or erratic environmental conditions.
3. It contributes to understanding the complex interactions between the molecular pathways for signalling in response to abiotic stress with those controlling cell and organ responses.
4. It will contribute to improve agricultural production, with a better adaptation to climate changes. It will allow to better exploit and develop agriculture in marginal lands and regions in Vietnam, particularly those already affected by adverse water or salinity stress conditions.
The perspectives of this work will involve collaboration with other institutions to develop research and enhanced breeding in rice. These will be based on state-of-the-art knowledge on physiological, cellular, molecular and genetic processes obtained on the plant model Arabidopsis and related to plant tolerance and adaptation to water stress.
References
1 Steudle, E. & Peterson, C. A. How does water get through roots? J. Exp. Bot. 49, 775-788 (1998).
2 Maurel, C., Verdoucq, L., Luu, D.-T. & Santoni, V. Plant aquaporins: membrane channels with multiple integrated functions. Annu Rev Plant Biol 59, 595-624 (2008).
3 Javot, H. et al. Role of a single aquaporin isoform in root water uptake. Plant Cell 15, 509-522 (2003).
4 Sakurai, J., Ishikawa, F., Yamaguchi, T., Uemura, M. & Maeshima, M. Identification of 33 rice aquaporin genes and analysis of their expression and function. Plant Cell Physiol 46, 1568-1577 (2005).
5 Sparkes, I. A., Runions, J., Kearns, A. & Hawes, C. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat. Protocols 1, 2019-2025 (2006).
6 Wang, Y. et al. OsRab5a regulates endomembrane organization and storage protein trafficking in rice endosperm cells. The Plant Journal 64, 812-824 (2010).
7 Boursiac, Y. et al. Early effects of salinity on water transport in Arabidopsis roots. Molecular and cellular features of aquaporin expression. Plant Physiol 139, 790-805 (2005).
8 Sallaud, C. et al. Highly efficient production and characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics. Theor Appl Genet 106, 1396-1408 (2003).
9 Barach, J., Clay, J., Raquet, B., Steiner, J. & Tolman, R. Field to Market: The Keystone Alliance for Sustainable Agriculture Environmental Resource Indicators for Measuring Outcomes of On-Farm Agricultural Production in the United States. (The Keystone Center, 2009).
Global changes are predicted to dramatically increase the variability of water supply, both in space and time, and will, thus affect crop yield. Several Mediterranean regions are already under severe risk of drought, extreme temperatures, and of other types of abiotic stress linked to water (e.g. as a result of periodic flooding, or salt stress). Southern countries such as Vietnam have also to face global changes. For instance, compared to the period 1980-2000, climate models predict an increase in temperature in the range of 1.2°C– 4.5°C in the North and in the range of 0.5°C – 3.0°C in the South and a sea level raise as high as 15 – 90 cm along the coastline of Vietnam, for 2070. Since Vietnam rice production is mainly located in the two deltas of Red river and Mekong, this sea level raise will be detrimental. Furthermore, in the Vietnam mountainous regions, the rice production relies essentially on rain fed watering. Changing the regime of watering is also detrimental. Plants are subjected to numerous abiotic stresses such as drought, salinity, extreme temperatures s, nutrient deficiencies and mineral toxicities, reducing their growth and therefore having a major impact on crop yield. Unfortunately, the adaptive mechanisms which allow some plants to cope with abiotic stresses are poorly understood. The control of water flow from the soil to the atmosphere via the root-to-stomata continuum is a central mechanism for plant growth and survival. More specifically, water transport in plant roots has been the object of intense research1. It was shown that water channel proteins named aquaporins mediate a significant part of the root hydraulic conductivity (Lpr) in numerous plant species including the model plant Arabidopsis thaliana and crops such as rice, maize, grapevine, or legumes2. Several of the above mentioned stresses are known to impact the water transport properties of plant tissues and to act on aquaporin regulation2.
ORYZAQUA project is willing to focus on rice (Oryza sativa) for understanding the function and regulation of aquaporins at the cellular level, using root cells of rice plantlets as a model to address the effects of salinity stress. Here, we want to focus on plantlets two weeks after germination, since the transplanting in the paddy fields is proceeded at this critical developmental stage when the plants are potentially subjected to severe stresses. The first aim is to address the water transport function of rice aquaporins in the root system. Next, we will monitor their subcellular localization by means of cell biology approaches.
Main results
Water transport function of aquaporins in the rice root system
We used a pressure-chamber system to monitor root hydraulic conductivity (Lpr)3. We compared two genotypes with contrasted watering regimes: Nipponbare is an irrigated lowland japonica rice and its genome sequence is now available; Azucena is a rainfed upland rice variety. Rice compared to Arabidopsis, presents a root architecture characterized by a fibrous system comprising four types of roots (the radicle, the crown roots, the large lateral roots, and the small lateral roots). These different types of roots are morphologically and anatomically distinct. To take into account the contribution of crown-root type, the genotype crl1, standing for crown-root-less mutant, was also analysed. Contrasted Lpr values were observed between genotypes (Figure 1). The two lowland and upland genotypes showed a marked difference. Interestingly, the mutant crl1 showed the lowest Lpr value. The salt-stress effect on the Lpr was also monitored. We observed a decrease of these values, indicating that salt has strong and fast inhibiting effects on the water transport capacities of rice plantlets.
Subcellular localization of rice aquaporins and sub-cellular markers
Here, we monitored the sub-cellular localization of selected rice aquaporins, the Plasma membrane Intrinsic Proteins (OsPIPs), by means of confocal laser scanning microscopy. For this, we cloned the cDNA sequences of four most highly root-expressed isoforms OsPIP1;1, OsPIP2;1, OsPIP2;4, and OsPIP2;54 and fused them individually with the sequence of the green fluorescent protein (GFP), under the control of a Cauliflower Mosaic Virus 35S promoter. The constructs were carried by a binary vector and were tested by the agroinfiltration method which allows a rapid observation of the localization of the transgenic products5. The constructs labelled the periphery of the cells indicating a plasma membrane (PM) localization, consistent with the nomenclature of these isoforms (Figure 2A-2E). OsRab5, a marker of the pre-vacuolar compartments (PVC)6 was expressed as a protein fusion with the fluorescent protein mCherry and showed a labelling surrounding some intracellular compartments such as small vacuoles (Figure 2F). We were also curious of the sub-cellular localization of the construct in Arabidopsis thaliana. For this, the floral-dip method was employed for obtaining Arabidopsis transgenic plants stably transformed and expressing individual OsPIP-GFP constructs (Figure 3). These constructs showed a peripheral labelling in agreement with the expected localization in the PM. Interestingly, under salt stress, a strong intracellular labelling was observed (Figure 3), reminiscent of what was observed for Arabidopsis PIP constructs in a previous study7. OsRab5-construct expression in Arabidopsis labelled intracellular compartments in control and salt-stressed plants (Figure 3). Finally, the Nipponbare genotype was stably transformed by Agrobacterium via a calli co-cultivation method8. Transgenic plants expressing OsPIP1;1-GFP or OsPIP2;1-GFP were obtained and the sub-cellular localization of OsPIP1;1-GFP was shown here as representative of the family members. In control conditions (Figure 4 upper panels), we observed a strict labelling of the PM for all the cells amenable to confocal microscopy analysis. Interestingly, the same pattern of localization was observed after a salt-stress (Figure 4 lower panels). We concluded that there is no detectable relocalization of OsPIP1;1-GFP construct upon 100 mM NaCl treatment.
Conclusion
This work establishes for the first time the strong inhibitory effect of salinity on the root water transport of rice plantlets. It is interpreted as a physiological mechanism to fastly (< 1 hour) respond to environmental challenges. Also, since this inhibitory effect is fast, it points to the regulation of aquaporin activity as a major factor of the whole-plant response to salinity. Interestingly, under salinity, we did not observe any differential localization of OsPIP1;1, taken here as a representative of PIP aquaporin isoforms. This would suggest a mechanism independent of PM aquaporin internalization to account for the reduction in Lpr.
Potential impact and use and any socio-economic impact
In the next 20 years, it is predicted that nearly half of the world will be living under severe water stress. We believe that reducing water needs through plant science technologies is feasible. Besides improved water management techniques, crops with improved water use efficiency may be developed. For example, a U.S. study found that fewer water is needed to grow irrigated cotton, corn, soybean and wheat today, compared to 20 years ago 9. Furthermore, developing drought tolerant crops is of importance and feasible (see this example of a corn tolerant to drought which is being developed for Africa: http://www.aatf-africa.org/wema/en/). ORYZAQUA will help enhance scientific knowledge on topics of crucial socio-economic importance. The agricultural production indeed needs to be better adapted to erratic, unpredictable environmental conditions that will arise under climate changes. This will allow the development of robust crops with improved traits for biomass yield, productivity and quality under adverse environmental conditions. Therefore, the issues that the proposal is intended to address are (1) increasing demand for plants for food uses and (2) interaction between climate change and crops.
ORYZAQUA will help establish major outputs and outcomes in several ways:
1. It addresses water stress affecting crop plants and will develop agricultural plant varieties better equipped to withstand water stress, useful for production of biomass and bioproducts.
2. It targets commercially important crops using state-of-the-art knowledge on physiological, molecular and genetic processes obtained on the model plant Arabidopsis. This knowledge, which relates to plant tolerance and adaptation to water stress, will be used for ultimately developing robust crops with improved traits for biomass yield, productivity and quality under adverse and/or erratic environmental conditions.
3. It contributes to understanding the complex interactions between the molecular pathways for signalling in response to abiotic stress with those controlling cell and organ responses.
4. It will contribute to improve agricultural production, with a better adaptation to climate changes. It will allow to better exploit and develop agriculture in marginal lands and regions in Vietnam, particularly those already affected by adverse water or salinity stress conditions.
The perspectives of this work will involve collaboration with other institutions to develop research and enhanced breeding in rice. These will be based on state-of-the-art knowledge on physiological, cellular, molecular and genetic processes obtained on the plant model Arabidopsis and related to plant tolerance and adaptation to water stress.
References
1 Steudle, E. & Peterson, C. A. How does water get through roots? J. Exp. Bot. 49, 775-788 (1998).
2 Maurel, C., Verdoucq, L., Luu, D.-T. & Santoni, V. Plant aquaporins: membrane channels with multiple integrated functions. Annu Rev Plant Biol 59, 595-624 (2008).
3 Javot, H. et al. Role of a single aquaporin isoform in root water uptake. Plant Cell 15, 509-522 (2003).
4 Sakurai, J., Ishikawa, F., Yamaguchi, T., Uemura, M. & Maeshima, M. Identification of 33 rice aquaporin genes and analysis of their expression and function. Plant Cell Physiol 46, 1568-1577 (2005).
5 Sparkes, I. A., Runions, J., Kearns, A. & Hawes, C. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat. Protocols 1, 2019-2025 (2006).
6 Wang, Y. et al. OsRab5a regulates endomembrane organization and storage protein trafficking in rice endosperm cells. The Plant Journal 64, 812-824 (2010).
7 Boursiac, Y. et al. Early effects of salinity on water transport in Arabidopsis roots. Molecular and cellular features of aquaporin expression. Plant Physiol 139, 790-805 (2005).
8 Sallaud, C. et al. Highly efficient production and characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics. Theor Appl Genet 106, 1396-1408 (2003).
9 Barach, J., Clay, J., Raquet, B., Steiner, J. & Tolman, R. Field to Market: The Keystone Alliance for Sustainable Agriculture Environmental Resource Indicators for Measuring Outcomes of On-Farm Agricultural Production in the United States. (The Keystone Center, 2009).
