Final Report Summary - REDOXHORMONE (Linking redox and hormonal signalling in Arabidopsis plants with altered levels of a stress-inducible glycosyltransferase)
1. Summary description of the project objectives
1.1 Assays for phenotype assessment, physiological and biochemical characterization in transgenic Arabidopsis plants overexpressing UGT74E2 and T-DNA knock out lines under different abiotic stress conditions
1.2 In vivo determination of hormone levels and glycosylated products
1.3 Molecular phenotyping of selected UGT74E2-KO lines
1.4 Nucleotide sequence analysis of the UGT74E2 promoter
1.5 Improved drought tolerance without undesired side effects in transgenic plants overexpressing UGT74E2
2. Description of the work performed since the beginning of the project
During this project, we demonstrate that the hydrogen peroxide-responsive UDP-glucosyltransferase UGT74E2 of Arabidopsis thaliana is involved in the modulation of plant architecture and water stress response through its activity toward the auxin indole-3-butyric acid (IBA). In UGT74E2 overexpressing transgenic plants, not only were IBA-glucose concentrations were increased, but also free IBA levels were elevated and the conjugated IAA pattern was modified. This altered IBA and IAA homeostasis was associated with architectural changes, including increased shoot branching and altered rosette shape, and resulted in significantly improved survival during drought and salt stress treatments. Hence, our results reveal that IBA and IBA-glucose are important regulators of morphological and physiological stress adaptation mechanisms, and provide molecular evidence for the interplay between hydrogen peroxide and auxin homeostasis through the action of an IBA UGT.
3. Description of the main results achieved so far
3.1 UGT74E2 perturbs auxin homeostasis
We have demonstrated that UGT74E2 is an IBA glucosyltransferase with a high and preferred activity toward IBA in in vitro assays and that its overexpression resulted in increased IBA-Glc levels in planta.
3.2 Modification of IBA homeostasis impinges on plant root and shoot development
Ectopic expression of UGT74E2 led to rounded shape of the rosette leaves, short petioles, dwarfed stature, and loss of apical dominance. Exogenously applied IBA or IBA-Glc could not mimic the architectural changes that are apparent in the gain-of-function lines, but decreased the leaf area and had an inhibitory effect on the flowering time. Therefore, the increased levels of IBA and IBA-Glc could explain the delayed flowering phenotype displayed by UGT74E2OE plants.
3.3 UGT74E2 is involved in stress-induced morphological adaptations
UGT74E2 overexpression not only affected the plant architecture, but also allowed transgenic plants to survive prolonged periods of drought and high salinity, suggesting a relationship between architectural changes and environmental stress resistance. Based on the phenotype and stress tolerance of UGT74E2OE plants and the stress inducibility of the gene, we can hypothesise that UGT74E2 is one component of the ROS signalling pathway that alters the auxin responsiveness, leading to stress-induced reorientation of growth, directly relevant for plant adaptation. In this sense, the tissue-specific production of UGT74E2 reveals a clear role of these IBA-modifying proteins in the auxin distribution. UGT74E2 is expressed at sites of auxin synthesis and where the hormone accumulates: lateral root primordia, root tips, primordial leaf tips, and hydathodes.
3.4 Auxins might assist in safeguarding the photosynthetic capacity during water stress
Our observations that IBA and IBA-Glc are strongly induced by osmotic stress and that constitutively increased levels of free and glucosylated IBA correlate with water stress tolerance imply a significant role of IBA in adaptation to water stress. Although we could mimic the photosynthetic response of UGT74E2OE plants during water stress by exogenously supplied IAA by using PEG-stressed wild-type seedlings, IBA and IBA-Glc accumulation failed to simulate the effects of stress on the photosynthetic parameters. These results suggest that the protective auxin-dependent response relies on an increase in IAA levels that is triggered by water stress signalling. Thus, photosynthesis, like so many other physiological processes, is apparently modulated by auxins, allowing plants to adjust or adapt their photosynthesis to environmental cues. However, further studies will be needed to clarify the role of IBA and IBA-Glc on photosynthetic processes under stress.
3.5 IBA is involved in water stress responses, independently of ABA
The water stress-tolerant phenotype of UGT74E2OE seems be a direct consequence of increased IBA and IBA-Glc levels rather than derived from an altered ABA homeostasis. Firstly, ABA and ABA-Glc levels were similar in wild-type and transgenic plants, both under control or dehydration conditions.
3.6 Auxins amplify ABA-induced inhibition of seed germination
Both germination and seedling growth were more sensitive to ABA in UGT74E2OE plants. At these developmental stages, crosstalk between ABA- and auxin-dependent responses has been reported to take place, in which either ABA-dependent repression of growth is potentiated by auxin (Liu et al., 2007) or auxin repression of embryonic axis elongation is enhanced by ABA (Belin et al., 2009). In this project we demonstrate that high levels of IBA, IBA-Glc, or IAA enhance the ABA inhibition of germination as well as of seedling development.
4. Conclusions
In summary, plant morphogenesis, including shoot branching, leaf area, and sprouting of axillary buds is affected by IBA and/or IBA-Glc homeostasis. Moreover, IBA-Glc can act as a physiologically active form of auxin on processes, such as rosette shape and flowering. Because of its spatial restriction to younger tissues, UGT74E2 might play a prominent role in stress-induced protective architectural changes in plants. The mechanisms that steer the IBA/IBA-Glc-dependent water stress-tolerant phenotype are less clear. Several lines of evidence hint at an action through improved osmotic adjustment or decreased water consumption rather than through a direct modulation of ABA-dependent protective responses. The correlation between the stress-inducible UGT74E2 expression and the enhanced auxin levels in newly formed tissues reflects a, previously undescribed, auxin-dependent mechanism to protect these tissues during water stress events.
5. Potential impact of the results
Clearly, manipulation of IBA homeostasis might be a new avenue in crop protection for water stress tolerance. In future research, the analysis of crosses between UGT74E2 overexpressors and mutants blocked in IBA biosynthesis, transport, and oxidation to IAA would allow us to investigate the timing and importance of IBA and IBA-to-IAA conversion in plant development, in addition to their role during water stress. We got interesting results about the possible role of IBA, IBA-Glc, and IAA on the control of photosynthesis as well as the protection of photosynthesis by increased IAA levels. These date open new and promising perspectives to investigate the role of auxin on photosynthesis and would lead to future tools to improve yields on crops.
In the light of a growing need for a more sustainable society, detailed understanding of plant responses to oxidative stress may result in novel strategies to improve yields of cultivated crops. Improved yields of cultivated crops, used for food and non-food applications, are highly desirable since the European ambitions regarding the future use of renewable resources, such as biofuels or bioplastics, will require an important increase in total biomass production.
References
Liu, P.-P. et al., (2007). Repression of auxin response factor10 by microRNA160 is critical for seed germination and post germination stages. Plant J. 52: 133 146.
Belin, C. et al., (2009). Abscisic acid represses growth of the Arabidopsis embryonic axis after germination by enhancing auxin signaling. Plant Cell 21: 2253-2268.
1.1 Assays for phenotype assessment, physiological and biochemical characterization in transgenic Arabidopsis plants overexpressing UGT74E2 and T-DNA knock out lines under different abiotic stress conditions
1.2 In vivo determination of hormone levels and glycosylated products
1.3 Molecular phenotyping of selected UGT74E2-KO lines
1.4 Nucleotide sequence analysis of the UGT74E2 promoter
1.5 Improved drought tolerance without undesired side effects in transgenic plants overexpressing UGT74E2
2. Description of the work performed since the beginning of the project
During this project, we demonstrate that the hydrogen peroxide-responsive UDP-glucosyltransferase UGT74E2 of Arabidopsis thaliana is involved in the modulation of plant architecture and water stress response through its activity toward the auxin indole-3-butyric acid (IBA). In UGT74E2 overexpressing transgenic plants, not only were IBA-glucose concentrations were increased, but also free IBA levels were elevated and the conjugated IAA pattern was modified. This altered IBA and IAA homeostasis was associated with architectural changes, including increased shoot branching and altered rosette shape, and resulted in significantly improved survival during drought and salt stress treatments. Hence, our results reveal that IBA and IBA-glucose are important regulators of morphological and physiological stress adaptation mechanisms, and provide molecular evidence for the interplay between hydrogen peroxide and auxin homeostasis through the action of an IBA UGT.
3. Description of the main results achieved so far
3.1 UGT74E2 perturbs auxin homeostasis
We have demonstrated that UGT74E2 is an IBA glucosyltransferase with a high and preferred activity toward IBA in in vitro assays and that its overexpression resulted in increased IBA-Glc levels in planta.
3.2 Modification of IBA homeostasis impinges on plant root and shoot development
Ectopic expression of UGT74E2 led to rounded shape of the rosette leaves, short petioles, dwarfed stature, and loss of apical dominance. Exogenously applied IBA or IBA-Glc could not mimic the architectural changes that are apparent in the gain-of-function lines, but decreased the leaf area and had an inhibitory effect on the flowering time. Therefore, the increased levels of IBA and IBA-Glc could explain the delayed flowering phenotype displayed by UGT74E2OE plants.
3.3 UGT74E2 is involved in stress-induced morphological adaptations
UGT74E2 overexpression not only affected the plant architecture, but also allowed transgenic plants to survive prolonged periods of drought and high salinity, suggesting a relationship between architectural changes and environmental stress resistance. Based on the phenotype and stress tolerance of UGT74E2OE plants and the stress inducibility of the gene, we can hypothesise that UGT74E2 is one component of the ROS signalling pathway that alters the auxin responsiveness, leading to stress-induced reorientation of growth, directly relevant for plant adaptation. In this sense, the tissue-specific production of UGT74E2 reveals a clear role of these IBA-modifying proteins in the auxin distribution. UGT74E2 is expressed at sites of auxin synthesis and where the hormone accumulates: lateral root primordia, root tips, primordial leaf tips, and hydathodes.
3.4 Auxins might assist in safeguarding the photosynthetic capacity during water stress
Our observations that IBA and IBA-Glc are strongly induced by osmotic stress and that constitutively increased levels of free and glucosylated IBA correlate with water stress tolerance imply a significant role of IBA in adaptation to water stress. Although we could mimic the photosynthetic response of UGT74E2OE plants during water stress by exogenously supplied IAA by using PEG-stressed wild-type seedlings, IBA and IBA-Glc accumulation failed to simulate the effects of stress on the photosynthetic parameters. These results suggest that the protective auxin-dependent response relies on an increase in IAA levels that is triggered by water stress signalling. Thus, photosynthesis, like so many other physiological processes, is apparently modulated by auxins, allowing plants to adjust or adapt their photosynthesis to environmental cues. However, further studies will be needed to clarify the role of IBA and IBA-Glc on photosynthetic processes under stress.
3.5 IBA is involved in water stress responses, independently of ABA
The water stress-tolerant phenotype of UGT74E2OE seems be a direct consequence of increased IBA and IBA-Glc levels rather than derived from an altered ABA homeostasis. Firstly, ABA and ABA-Glc levels were similar in wild-type and transgenic plants, both under control or dehydration conditions.
3.6 Auxins amplify ABA-induced inhibition of seed germination
Both germination and seedling growth were more sensitive to ABA in UGT74E2OE plants. At these developmental stages, crosstalk between ABA- and auxin-dependent responses has been reported to take place, in which either ABA-dependent repression of growth is potentiated by auxin (Liu et al., 2007) or auxin repression of embryonic axis elongation is enhanced by ABA (Belin et al., 2009). In this project we demonstrate that high levels of IBA, IBA-Glc, or IAA enhance the ABA inhibition of germination as well as of seedling development.
4. Conclusions
In summary, plant morphogenesis, including shoot branching, leaf area, and sprouting of axillary buds is affected by IBA and/or IBA-Glc homeostasis. Moreover, IBA-Glc can act as a physiologically active form of auxin on processes, such as rosette shape and flowering. Because of its spatial restriction to younger tissues, UGT74E2 might play a prominent role in stress-induced protective architectural changes in plants. The mechanisms that steer the IBA/IBA-Glc-dependent water stress-tolerant phenotype are less clear. Several lines of evidence hint at an action through improved osmotic adjustment or decreased water consumption rather than through a direct modulation of ABA-dependent protective responses. The correlation between the stress-inducible UGT74E2 expression and the enhanced auxin levels in newly formed tissues reflects a, previously undescribed, auxin-dependent mechanism to protect these tissues during water stress events.
5. Potential impact of the results
Clearly, manipulation of IBA homeostasis might be a new avenue in crop protection for water stress tolerance. In future research, the analysis of crosses between UGT74E2 overexpressors and mutants blocked in IBA biosynthesis, transport, and oxidation to IAA would allow us to investigate the timing and importance of IBA and IBA-to-IAA conversion in plant development, in addition to their role during water stress. We got interesting results about the possible role of IBA, IBA-Glc, and IAA on the control of photosynthesis as well as the protection of photosynthesis by increased IAA levels. These date open new and promising perspectives to investigate the role of auxin on photosynthesis and would lead to future tools to improve yields on crops.
In the light of a growing need for a more sustainable society, detailed understanding of plant responses to oxidative stress may result in novel strategies to improve yields of cultivated crops. Improved yields of cultivated crops, used for food and non-food applications, are highly desirable since the European ambitions regarding the future use of renewable resources, such as biofuels or bioplastics, will require an important increase in total biomass production.
References
Liu, P.-P. et al., (2007). Repression of auxin response factor10 by microRNA160 is critical for seed germination and post germination stages. Plant J. 52: 133 146.
Belin, C. et al., (2009). Abscisic acid represses growth of the Arabidopsis embryonic axis after germination by enhancing auxin signaling. Plant Cell 21: 2253-2268.
