Final Report Summary - ETSIGDEV (Role of EIN3 and ethylene signaling in Arabidopsis development)
Project context and objectives
Hormones play a critical role throughout the life-cycle of plants. The gas ethylene is, together with auxin, one of the earliest characterised plant hormones. Ethylene distinguishes itself among the other plant hormones by its simple hydrocarbon structure (C2H4) and its gaseous nature. This simple molecule, however, plays a major role in plant growth and development by influencing a wide range of complex physiological processes throughout the entire plant life-cycle, from seed germination to flowering, fruit ripening and senescence. Ethylene signalling begins with ethylene binding to, and inactivating a family of, ethylene receptors. In the absence of ethylene, these receptors activate CTR1, a MAPKKK (mitogen activating protein kinase kinase kinase) that negatively regulates the pathway. After inactivation of CTR1, EIN2 promotes ethylene responses via the downstream transcription factor EIN3. EIN3 activates primary targets of the ethylene response cascade, such as ERF1. In the absence of ethylene stress, EIN3 protein is constitutively subjected to proteasomal degradation by two related ubiquitin ligases, SCFEBF1 and SCFEBF2. ebf1 and ebf2 are F-box proteins that act as the substrate recognition components of SCFEBF1/2. It is only in the presence of ethylene, or in plants that are mutant for ebf1 and ebf2, that EIN3 is protected from degradation and can fulfil its role in the ethylene-signalling cascade.
We have previously identified ebf1ebf2 hypomorphic double mutants, which show constitutive over-accumulation of EIN3 protein, and sport several strong developmental defects such as pronounced dwarfism, abnormally small flowers with protruding gynoecium, rosettes with small-sized leaves and male sterility. In addition, we and others found that ebf1ebf2 double null mutants are even more strongly affected as they are seedling lethal. This seedling lethality is not seen in other constitutive ethylene response mutants, such as ctr1.
In this project, we investigated ebf1ebf2 double knockouts in detail using different approaches. Originally three tasks were identified:
1. the identification of EIN3 primary targets;
2. the role of EIN3 in cell differentiation and cell fate;
3. the role EIN3 in crosstalk between ethylene and auxin signalling.
Work performed
As described in the mid-term report, the identification of primary EIN3 targets by ChIP on Chip or equivalent methods was aborted because at the start of the project other groups published several direct EIN3 targets. This allowed for the identification of EIN3 binding sites in candidate promoters by bio-informatics and there is no need for experimental identification of EIN3 targets by global 'omics' methods. In addition, Joseph R. Ecker presented a nearly complete identification of EIN3 targets at a plenary session talk at the 2009 International Conference on Arabidopsis Research in Edinburgh.
Tasks 2 and 3 are closely linked and will be therefore discussed together.
We found that ebf1ebf2 double knockout mutants appear to be normal during embryo development but have a reduced meristem post germination. The root meristem gradually deteriorates and collapses as the plants die. This effect is dependent on EIN3, as ebf1ebf2ein3 triple mutants are essentially normal. Introgression of markers for cell differentiation indicated that prior to death differentiation processes occur in ebf1ebf2 double-knockout lines.
Many ethylene dependent processes occur through changes in auxin flux or local auxin biosynthesis. As ethylene has been shown to restrict root growth largely through inhibition of cell elongation which occurs in postmeristematic cells and not in the root meristem, and auxin is a well-known mobile signal in plants, auxin would be plausible as a mediator of EIN3-dependent root meristem shrinking. To investigate whether meristem collapse and seedling death of ebf1ebf2 double-knockout seedlings can be attenuated by reducing auxin flux or by blocking auxin biosynthesis, we generated pin2ebf1ebf2 triple mutants and treated ebf1ebf2 seedlings with an anti-auxin. Preliminary results suggest that pin2ebf1ebf2 plants are phenotypically similar to ebf1ebf2 plants; they still show meristem collapse and seedling lethality. Likewise, treatment of ebf1ebf2 seedlings with the anti-auxin clofibric acid neither rescued the meristem size nor did it restore seedling viability.
To test if the meristem collapse in ebf1ebf2 double-knockout lines is a local effect caused by EIN3 stabilisation in the meristem, we constructed genomic fusions of EIN3 to a triple GFP reporter. We found that EIN3-3xGFP is stabilised by ethylene (as is native EIN3 protein) and that EIN3 3xGFP is accumulating in high levels in ethylene-treated plants and in ebf1ebf2 knockout lines, including the root meristem. In addition, we found that ebf1ebf2 mutant embryos are phenotypically normal and do not show EIN3 protein accumulation. However, after germination, EIN3 protein is abundant in ebf1ebf mutant plants or after ethylene treatment of wild type plants. Thus EIN3 protein accumulation is a phenomene that occurs after germination, which explains the noram appearance of ebf1ebf2 mutant embryos.
In addition, we showed that ectopic expression of ebf2 under the control of the meristem specific promoter RCH1 at least partially rescued the root meristem defect of ebf1ebf2 knockout plants. Taken together, these results indicate that the stabilisation of EIN3 in ebf1ebf2 knockout lines causes root meristem shrinkage and finally root meristem collapse. This process is a direct consequence of EIN3 accumulation in the meristem.
Auxin does not appear to play a major role in this process, and the signalling events downstream of EIN3 that cause root meristem collapse and seedling death remain to be identified.
This was a basic research project, driven by curiosity and has no socio-economic impact.
Contact details for any inquiries:
Thomas Potuschak (scientist in charge)
E-mail:
Thomas.potuschak@ibmp-cnrs.unistra.fr
Address:
IBMP-CNRS, 12 rue du général Zimmer, 67084 Strasbourg Cedex, France.
Hormones play a critical role throughout the life-cycle of plants. The gas ethylene is, together with auxin, one of the earliest characterised plant hormones. Ethylene distinguishes itself among the other plant hormones by its simple hydrocarbon structure (C2H4) and its gaseous nature. This simple molecule, however, plays a major role in plant growth and development by influencing a wide range of complex physiological processes throughout the entire plant life-cycle, from seed germination to flowering, fruit ripening and senescence. Ethylene signalling begins with ethylene binding to, and inactivating a family of, ethylene receptors. In the absence of ethylene, these receptors activate CTR1, a MAPKKK (mitogen activating protein kinase kinase kinase) that negatively regulates the pathway. After inactivation of CTR1, EIN2 promotes ethylene responses via the downstream transcription factor EIN3. EIN3 activates primary targets of the ethylene response cascade, such as ERF1. In the absence of ethylene stress, EIN3 protein is constitutively subjected to proteasomal degradation by two related ubiquitin ligases, SCFEBF1 and SCFEBF2. ebf1 and ebf2 are F-box proteins that act as the substrate recognition components of SCFEBF1/2. It is only in the presence of ethylene, or in plants that are mutant for ebf1 and ebf2, that EIN3 is protected from degradation and can fulfil its role in the ethylene-signalling cascade.
We have previously identified ebf1ebf2 hypomorphic double mutants, which show constitutive over-accumulation of EIN3 protein, and sport several strong developmental defects such as pronounced dwarfism, abnormally small flowers with protruding gynoecium, rosettes with small-sized leaves and male sterility. In addition, we and others found that ebf1ebf2 double null mutants are even more strongly affected as they are seedling lethal. This seedling lethality is not seen in other constitutive ethylene response mutants, such as ctr1.
In this project, we investigated ebf1ebf2 double knockouts in detail using different approaches. Originally three tasks were identified:
1. the identification of EIN3 primary targets;
2. the role of EIN3 in cell differentiation and cell fate;
3. the role EIN3 in crosstalk between ethylene and auxin signalling.
Work performed
As described in the mid-term report, the identification of primary EIN3 targets by ChIP on Chip or equivalent methods was aborted because at the start of the project other groups published several direct EIN3 targets. This allowed for the identification of EIN3 binding sites in candidate promoters by bio-informatics and there is no need for experimental identification of EIN3 targets by global 'omics' methods. In addition, Joseph R. Ecker presented a nearly complete identification of EIN3 targets at a plenary session talk at the 2009 International Conference on Arabidopsis Research in Edinburgh.
Tasks 2 and 3 are closely linked and will be therefore discussed together.
We found that ebf1ebf2 double knockout mutants appear to be normal during embryo development but have a reduced meristem post germination. The root meristem gradually deteriorates and collapses as the plants die. This effect is dependent on EIN3, as ebf1ebf2ein3 triple mutants are essentially normal. Introgression of markers for cell differentiation indicated that prior to death differentiation processes occur in ebf1ebf2 double-knockout lines.
Many ethylene dependent processes occur through changes in auxin flux or local auxin biosynthesis. As ethylene has been shown to restrict root growth largely through inhibition of cell elongation which occurs in postmeristematic cells and not in the root meristem, and auxin is a well-known mobile signal in plants, auxin would be plausible as a mediator of EIN3-dependent root meristem shrinking. To investigate whether meristem collapse and seedling death of ebf1ebf2 double-knockout seedlings can be attenuated by reducing auxin flux or by blocking auxin biosynthesis, we generated pin2ebf1ebf2 triple mutants and treated ebf1ebf2 seedlings with an anti-auxin. Preliminary results suggest that pin2ebf1ebf2 plants are phenotypically similar to ebf1ebf2 plants; they still show meristem collapse and seedling lethality. Likewise, treatment of ebf1ebf2 seedlings with the anti-auxin clofibric acid neither rescued the meristem size nor did it restore seedling viability.
To test if the meristem collapse in ebf1ebf2 double-knockout lines is a local effect caused by EIN3 stabilisation in the meristem, we constructed genomic fusions of EIN3 to a triple GFP reporter. We found that EIN3-3xGFP is stabilised by ethylene (as is native EIN3 protein) and that EIN3 3xGFP is accumulating in high levels in ethylene-treated plants and in ebf1ebf2 knockout lines, including the root meristem. In addition, we found that ebf1ebf2 mutant embryos are phenotypically normal and do not show EIN3 protein accumulation. However, after germination, EIN3 protein is abundant in ebf1ebf mutant plants or after ethylene treatment of wild type plants. Thus EIN3 protein accumulation is a phenomene that occurs after germination, which explains the noram appearance of ebf1ebf2 mutant embryos.
In addition, we showed that ectopic expression of ebf2 under the control of the meristem specific promoter RCH1 at least partially rescued the root meristem defect of ebf1ebf2 knockout plants. Taken together, these results indicate that the stabilisation of EIN3 in ebf1ebf2 knockout lines causes root meristem shrinkage and finally root meristem collapse. This process is a direct consequence of EIN3 accumulation in the meristem.
Auxin does not appear to play a major role in this process, and the signalling events downstream of EIN3 that cause root meristem collapse and seedling death remain to be identified.
This was a basic research project, driven by curiosity and has no socio-economic impact.
Contact details for any inquiries:
Thomas Potuschak (scientist in charge)
E-mail:
Thomas.potuschak@ibmp-cnrs.unistra.fr
Address:
IBMP-CNRS, 12 rue du général Zimmer, 67084 Strasbourg Cedex, France.