Final Report Summary - HYDROTROPISM (Abscisic acid and the mechanisms that regulate hydrotropism.)
Water availability is one of the major factors limiting plants’ survival. Drought, as one of the main abiotic stresses, affects plant growth and development and causes economic and environmental damages worldwide. Among the different adaptive responses that plants activate under water stress the regulation of root growth can be modified in order to increase water uptake. This project focuses on the study of hydrotropism, a mechanism that guides root growth away from regions with low water potential under water limiting conditions. This response was described in the XIX century by Darwin and Sachs (1, 2). However, the mechanisms driving hydrotropism are still poorly understood.
Abscisic acid (ABA) is the main plant hormone regulating adaptive responses to abiotic stress. The molecular mechanisms by which ABA triggers stomatal closure or modification of gene expression under water deficits have been described in detail. By contrast, for hydrotropism, some results suggest the role of ABA in this process but the direct relation between ABA and root hydrotropic bending still needs to be elucidated. Within this context, the aim of this project was to clarify the relation between ABA and hydrotropism. ABA responsive genes are up-regulated after the application of a moisture gradient (3). Besides, some components of ABA signalling and biosynthesis are important for the hydrotropic response as their respective knock out mutants show impaired hydrotropic phenotypes. These include Sucrose non-fermenting-1 related protein kinase 2 (SnRK2) (D. Dietrich unpublished results), the protein serine/threonine phosphatases 2C (PP2C) (4) and zeaxanthin epoxidase ABA1 (5). We have generated translational fusions of two SnRK2s kinases (SnRK2.2 and SnRk2.3) in order to identify the important tissues in the root for hydrotropism. Both are present in the root tip and in the elongation zone which is in accordance with previous studies reporting the importance of root cap for moisture perception (Fig. 1A).
We hypothesized that an ABA gradient between the two sides of the root could be the signal causing this differential growth. An ABA gradient could be formed either by transporting ABA towards a part of the root or by locally produce ABA in the area where the water potential is lower. The ABA biosynthetic pathway has been precisely characterized in the past. In fact, the ABA-deficient mutant, aba1 shows a reduced hydrotropic response. Several ABA transporters have been isolated in the last years but further information is needed to understand how ABA is transported between tissues and organs. By using a split agar-based plates that contain two media with different water potential it is possible to measure the hydrotropic response as an estimation of the root curvature (Fig. 1B). Using this system we have analysed this response in mutants affected in ABA transport or biosynthesis. We have screened the hydrotropic phenotype of knock out mutants of the ABA transporters such as abcg25, abcg40 and members of the transporter family NRT1/PTR and of ABA biosynthetic genes such as nced2nced3. However, single or double mutants show responses similar to wild type. These results show that only part of the ABA machinery could participate in the response and they also highlight that genetic redundancy could mask the phenotypes in families of genes with a high number of members such as the NCED family.
The identification of new targets is essential to understand better this process. We have performed an RNAseq experiment using RNA extracted from hydrotropically stimulated root tips of seedlings from wild type and compared to the same material from the hydrotropic response mutant snrk2.2snrk2.3. The differentially expressed genes can be now analysed using our split agar-based system in order to check if they show hydrotropic defects.
With the aim of study if an asymmetric distribution of ABA triggers root bending under an osmotic gradient we have used ABA reporters to monitor ABA distribution in the root. We have monitored ABA distribution during root bending under hydrotropic stimulation using ABA transcriptional reporters and we have observed that bending starts within the first hour after applying an osmotic gradient. This fact means that changes in ABA must be detected in this interval of time and implies that ABA transcriptional reporters may not be adequate as they require hours to show significant inductions (Fig. 1C). The distribution and accumulation of ABA in the plant has been investigated for years but no direct mechanisms of detection in vivo had been described when this project started. In collaboration with Prof. W.Frommer (Carnegie Institution for Science https://dpb.carnegiescience.edu/) we have used FRET-based ABA reporters to monitor ABA abundance and distribution during this response (6). This first generation of FRET-based ABA sensors detect changes in ABA concentration within minutes but they require high concentrations of the hormone to be activated. The fact that during hydrotropic stimulation we haven’t observed significant differences using these sensors could be due to the fact that changes on ABA concentration might be under the level of the detection of these reporters. We are working on the improvement of these sensors so that they can detect lower changes in ABA, more according to the scale observed in natural stress conditions.
In parallel to molecular approaches we were also interested into study how roots respond to drought within their natural soil environment. The University of Nottingham has a new ERC funded X-ray computed tomography (CT) facility for the study of root systems in soil. We have used CT imaging to visualize how roots growth towards parts of the soil with higher water content (Fig. 1E). In addition, in collaboration with J.Dinneny we have used the GLO-Roots facility to analyse the expression of water stress reporters in soil under drought conditions (Fig. 1D). These two soil approaches can be crucial to show how the whole root system responds to water deficit.
Roots are exposed to multiple external signals that need to be integrated in order to adapt to environmental changes. The hydrotropic response is a complex process regulated by ABA but also by other factors. Approaches directed to isolate new downstream targets like the one performed in this study using RNAseq can help to clarify the relations between the different pathways participating in the hydrotropic response. In addition, the challenging task of generating better ABA sensors with high affinity will help to clarify the ABA role during the hydrotropic response. All the progress made during this project and the further results that will be generated will increase the knowledge in the way plants adapt to drought. Since the amount of regions affected by drought increase every year, these findings can have direct impact into improving crop productivity in areas where water is a limiting factor.
1. Sachs, J. in Arbeiten des botanischen Instituts in Wuerzburg. 209-222 (Wilhelm Engelmann, Leipzig, 1872).
2. Darwin, C. & Darwin, F. in The Power of Movements in Plants. Ch. 3, 180-186 (John Murray, 1880).
3. Moriwaki, T., Miyazawa, Y., Fujii, N., Takahashi, H. Light and abscisic acid signaling are integrated by MIZ1 gene expression and regulate hydrotropic response in roots of Arabidopsis thaliana. (2012). Plant, Cell & Environment 35, 1359-1368.
4. Antoni R, Gonzalez-Guzman M, Rodriguez L, Peirats-Llobet M, Pizzio GA, Fernandez MA, De Winne N, De Jaeger G, Dietrich D, Bennett MJ & Rodriguez PL (2013) PYRABACTIN RESISTANCE1-LIKE8 plays an important role for the regulation of abscisic acid signaling in root. Plant Physiol. 161: 931–41.
5. Takahashi, N., Goto, N., Okada, K. & Takahashi, H. Hydrotropism in abscisic acid, wavy, and gravitropic mutants of Arabidopsis thaliana. (2002). Planta 216, 203-211.
6. Jones, A. M., Danielson, J., ManojKumar, S. N., Lanquar, V., Grossmann, G. & Frommer, W. B. (2014). Abscisic acid dynamics in roots detected with genetically encoded FRET sensors. eLife Sciences 3.
Abscisic acid (ABA) is the main plant hormone regulating adaptive responses to abiotic stress. The molecular mechanisms by which ABA triggers stomatal closure or modification of gene expression under water deficits have been described in detail. By contrast, for hydrotropism, some results suggest the role of ABA in this process but the direct relation between ABA and root hydrotropic bending still needs to be elucidated. Within this context, the aim of this project was to clarify the relation between ABA and hydrotropism. ABA responsive genes are up-regulated after the application of a moisture gradient (3). Besides, some components of ABA signalling and biosynthesis are important for the hydrotropic response as their respective knock out mutants show impaired hydrotropic phenotypes. These include Sucrose non-fermenting-1 related protein kinase 2 (SnRK2) (D. Dietrich unpublished results), the protein serine/threonine phosphatases 2C (PP2C) (4) and zeaxanthin epoxidase ABA1 (5). We have generated translational fusions of two SnRK2s kinases (SnRK2.2 and SnRk2.3) in order to identify the important tissues in the root for hydrotropism. Both are present in the root tip and in the elongation zone which is in accordance with previous studies reporting the importance of root cap for moisture perception (Fig. 1A).
We hypothesized that an ABA gradient between the two sides of the root could be the signal causing this differential growth. An ABA gradient could be formed either by transporting ABA towards a part of the root or by locally produce ABA in the area where the water potential is lower. The ABA biosynthetic pathway has been precisely characterized in the past. In fact, the ABA-deficient mutant, aba1 shows a reduced hydrotropic response. Several ABA transporters have been isolated in the last years but further information is needed to understand how ABA is transported between tissues and organs. By using a split agar-based plates that contain two media with different water potential it is possible to measure the hydrotropic response as an estimation of the root curvature (Fig. 1B). Using this system we have analysed this response in mutants affected in ABA transport or biosynthesis. We have screened the hydrotropic phenotype of knock out mutants of the ABA transporters such as abcg25, abcg40 and members of the transporter family NRT1/PTR and of ABA biosynthetic genes such as nced2nced3. However, single or double mutants show responses similar to wild type. These results show that only part of the ABA machinery could participate in the response and they also highlight that genetic redundancy could mask the phenotypes in families of genes with a high number of members such as the NCED family.
The identification of new targets is essential to understand better this process. We have performed an RNAseq experiment using RNA extracted from hydrotropically stimulated root tips of seedlings from wild type and compared to the same material from the hydrotropic response mutant snrk2.2snrk2.3. The differentially expressed genes can be now analysed using our split agar-based system in order to check if they show hydrotropic defects.
With the aim of study if an asymmetric distribution of ABA triggers root bending under an osmotic gradient we have used ABA reporters to monitor ABA distribution in the root. We have monitored ABA distribution during root bending under hydrotropic stimulation using ABA transcriptional reporters and we have observed that bending starts within the first hour after applying an osmotic gradient. This fact means that changes in ABA must be detected in this interval of time and implies that ABA transcriptional reporters may not be adequate as they require hours to show significant inductions (Fig. 1C). The distribution and accumulation of ABA in the plant has been investigated for years but no direct mechanisms of detection in vivo had been described when this project started. In collaboration with Prof. W.Frommer (Carnegie Institution for Science https://dpb.carnegiescience.edu/) we have used FRET-based ABA reporters to monitor ABA abundance and distribution during this response (6). This first generation of FRET-based ABA sensors detect changes in ABA concentration within minutes but they require high concentrations of the hormone to be activated. The fact that during hydrotropic stimulation we haven’t observed significant differences using these sensors could be due to the fact that changes on ABA concentration might be under the level of the detection of these reporters. We are working on the improvement of these sensors so that they can detect lower changes in ABA, more according to the scale observed in natural stress conditions.
In parallel to molecular approaches we were also interested into study how roots respond to drought within their natural soil environment. The University of Nottingham has a new ERC funded X-ray computed tomography (CT) facility for the study of root systems in soil. We have used CT imaging to visualize how roots growth towards parts of the soil with higher water content (Fig. 1E). In addition, in collaboration with J.Dinneny we have used the GLO-Roots facility to analyse the expression of water stress reporters in soil under drought conditions (Fig. 1D). These two soil approaches can be crucial to show how the whole root system responds to water deficit.
Roots are exposed to multiple external signals that need to be integrated in order to adapt to environmental changes. The hydrotropic response is a complex process regulated by ABA but also by other factors. Approaches directed to isolate new downstream targets like the one performed in this study using RNAseq can help to clarify the relations between the different pathways participating in the hydrotropic response. In addition, the challenging task of generating better ABA sensors with high affinity will help to clarify the ABA role during the hydrotropic response. All the progress made during this project and the further results that will be generated will increase the knowledge in the way plants adapt to drought. Since the amount of regions affected by drought increase every year, these findings can have direct impact into improving crop productivity in areas where water is a limiting factor.
1. Sachs, J. in Arbeiten des botanischen Instituts in Wuerzburg. 209-222 (Wilhelm Engelmann, Leipzig, 1872).
2. Darwin, C. & Darwin, F. in The Power of Movements in Plants. Ch. 3, 180-186 (John Murray, 1880).
3. Moriwaki, T., Miyazawa, Y., Fujii, N., Takahashi, H. Light and abscisic acid signaling are integrated by MIZ1 gene expression and regulate hydrotropic response in roots of Arabidopsis thaliana. (2012). Plant, Cell & Environment 35, 1359-1368.
4. Antoni R, Gonzalez-Guzman M, Rodriguez L, Peirats-Llobet M, Pizzio GA, Fernandez MA, De Winne N, De Jaeger G, Dietrich D, Bennett MJ & Rodriguez PL (2013) PYRABACTIN RESISTANCE1-LIKE8 plays an important role for the regulation of abscisic acid signaling in root. Plant Physiol. 161: 931–41.
5. Takahashi, N., Goto, N., Okada, K. & Takahashi, H. Hydrotropism in abscisic acid, wavy, and gravitropic mutants of Arabidopsis thaliana. (2002). Planta 216, 203-211.
6. Jones, A. M., Danielson, J., ManojKumar, S. N., Lanquar, V., Grossmann, G. & Frommer, W. B. (2014). Abscisic acid dynamics in roots detected with genetically encoded FRET sensors. eLife Sciences 3.
