Progress 08/01/01 to 07/31/05
Outputs
Biochemical, physiological, and molecular-genetic studies were done to investigate the role of EARLI1 in flowering time and the cold acclimation process of Arabidopsis. As proposed in Specific Aim 1a, different EARLI1 overexpression or RNAi knockdown lines were generated and their flowering time analyzed. Manipulating EARLI1 levels in a late-flowering Arabidopsis background had no clear effect on flowering time. Analysis of EARLI1 expression profiles in different flowering time mutants showed that the gene was differentially expressed in some mutants, however, no correlation within the same class of mutants was observed. Taken together, the results were not in agreement with the hypothesis that EARLI1 has a significant effect on flowering time and were not pursued further. Specifically, it was not tested whether EARLI1 was regulated by genes of different flowering pathways, as proposed as part of Specific Aim 1a. By contrast, manipulating EARLI1 levels had a
reproducible effect on the freezing tolerance of transgenic Arabidopsis plants. This was determined, as proposed in Specific Aim 1b, by measuring the survival rate of plants frozen at different subzero temperatures, or by measuring the cellular damage done to leaf tissues of frozen plants. The results indicated that EARLI1 protein partially protects plants against freezing stress. Because of this we shifted our project to functionally analyze EARLI1 using biochemical and molecular-genetic approaches not described in the grant proposal. We determined that the cold induction of EARLI1 was calcium and light dependent, but independent of the stress hormone abscisic acid. We generated antibodies to study the protein and determined that it forms higher order complexes through disulfide bonds. We also showed that the protein is hydrophobic or prone to aggregate in solution, which suggests that it is not a lipid transfer protein as previously assumed. We separated the proline-rich N-terminal
domain (PRD) from the 8-cystein-motif (8CM) C-terminus and showed that the PRD by itself was hydrophilic and soluble and that the 8CM was hydrophobic and insoluble. We used fusion proteins with GFP and did immunohistochemistry/confocal microscopy and cell fractionation experiments to show that EARLI1 is secreted into the extracellular space. Our current hypothesis is that its hydrophobic 8CM interacts with the outside part of the cell membrane and the hydrophilic PRP domain interacts with the cell wall. The secretion into the cell wall/plasma membrane space is thought to protect plant cells against environmental stresses. Because of this shift in focus some approaches described in Specific Aim 2a/b were not finished. However, we considered the biochemical analysis of the protein more important for elucidating the function of EARLI1. We have a new manuscript summarizing the biochemical and physiological analysis described in this report submitted to Plant Mol Biol. We also used some of
the funds to finish flowering time projects previously funded through USDA. One manuscript will be published in December 2006 in Plant Physiology, and a second one is being revised for resubmission to Plant Mol Biol.
Impacts
Secretion of proteins belonging to the EARLI1 gene family into the extracellular space helps to protect plant cells against environmental stresses such as freezing, and potentially drought or salt. Detailed information about the physiological and biochemical function of these genes will allow for the engineering of crop plants with increased stress tolerance.
Publications
- Bubier J and Schlappi M (2004) Cold induction of EARLI1, a putative Arabidopsis lipid transfer protein, is light and calcium dependent. Plant Cell & Environment 27: 929-936.
- Pan Y and Schlappi M (2006) Additive floral repression in Arabidopsis thaliana by FLC and its homologs MAF2 and MAF3. Plant Mol Biol (in revision).
- Schlappi MR (2006) FRIGIDA LIKE 2 is a functional allele in Landsberg erecta and compensates for a nonsense allele of FRIGIDA LIKE 1. Plant Physiol 142: in press (Dec. 2006).
- Zhang Y and Schlappi M (2006) The cold responsive hybrid proline-rich protein EARLI1 from Arabidopsis is localized to the cell periphery and forms higher order protein complexes via disulfide bonds. Plant Mol Biol (in review).
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Progress 10/01/03 to 09/30/04
Outputs
A main objective during this funding period was to determine whether Arabidopsis plants lacking EARLI1 mRNA suffered greater freezing damage after cold acclimation than wild-type plants. To this end we analyzed the freezing tolerance of Arabidopsis lines containing an RNAi construct against the putative LTP-coding region of EARLI1. RNA gel blot results demonstrated that after cold acclimation, RNAi-containing lines had no measurable amount of EARLI1 mRNA. We have additional preliminary results using protein gel blot analyses that RNAi-containing plants do not have detectable amounts of EARLI1 protein, even after 10 or 20 days of cold acclimation. Whole plant electrolyte leakage assays showed that plants lacking EARLI1 mRNA consistently leaked more ions between minus 6 degree C and minus 12 degree C than wild-type plants, indicating that they suffered a grater degree of freezing-induced cellular damage. These results, together with our published evidence that
overexpression of EARLI1 cDNA partially protects plants from freezing-induced cellular damage suggest that EARLI1 has a role in the cold acclimation response of Arabidopsis. Another objective during this funding period was to identify cold-specific response elements in the EARLI1 promoter. Different 5' and 3' deletions of the EARLI1 promoter were fused to the reporter gene GUS. The EARLI1 promoter-GUS constructs were introduced into plants and analyzed in the second year. We identified a 100 bp fragment conferring cold responsiveness to a minimal EARLI1 promoter-GUS construct. The fragment has several putative MYC and one putative MYB binding site. Site-directed mutagenesis during the fourth year of this funding period will narrow down the cold responsive cis-element. The same deletion constructs will be used in to identify regulatory elements for the light- and/or light/sugar responsiveness of EARLI1.
Impacts
Plants are sessile and vulnerable to environmental changes. Many plant genes are thus under environmental control. We are investigating the gene EARLI1, which is activated by cold temperatures, is light/sugar-responsive, and plays a role in the cold acclimation response of Arabidopsis, possibly by modifying the plasma composition during cold treatment. Further analysis of the role and regulation of EARLI1 will contribute to our understanding of how plants adapt to their environment and will provide basic knowledge for crop improvement.
Publications
- Bubier J and Schlappi M (2004) Cold induction of EARLI1, a putative Arabidopsis lipid transfer protein, is light and calcium dependent. Plant Cell & Environment 27, 929-936.
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Progress 10/01/02 to 09/30/03
Outputs
Our main objective during this funding period was to determine whether EARLI1 has a role in either flowering time and/or cold acclimation of Arabidopsis. EARLI1 is stably activated after vernalization in an FLC-containing, late-flowering mixed Columbia/Niederzenz background of Arabidopsis. We could show that FLC did not repress EARLI1 and that overexpression of the gene did not affect flowering time of transgenic plants. Moreover, EARLI1 was not vernalization-responsive in the pure Columbia background, suggesting that modifier genes in the mixed Columbia/Niederzenz background are responsible for vernalization responsiveness. We concluded that EARLI1 has a minor, if any, role in the vernalization response. By contrast, overexpression of EARLI1 protected non-cold-acclimated Arabidopsis plants from freezing-induced damage. This suggested that EARLI1 has a role in the cold acclimation response of Arabidopsis.
We also determined that the cold responsiveness of EARLI1 is light- and photoperiod-dependent. This is intriguing because light is critical for cold acclimation of plants. We further showed that cold-responsive expression of EARLI1 is ABA-independent but calcium-dependent. Another objective during this funding period was to identify environment-specific response elements in the promoter of EARLI1. Different deletions of the EARLI1 promoter were fused to the reporter gene GUS. The EARLI1 promoter-GUS constructs were introduced into plants and analyzed in the second year. We have identified a 100 bp fragment conferring cold responsiveness to a minimal EARLI1 promoter-GUS construct. The fragment has several putative MYC and one putative MYB binding site. Site-directed mutatagenesis during the third year of this funding period will identify cold responsive cis-elements. The same deletion constructs will be used in the third year to identify regulatory
elements for the light- and/or long-day-photoperiod responsiveness of EARLI1. These elements together with the identification of interaction proteins will allow us in future experiments to manipulate the environmental regulation of important crop plants.
Impacts
Plants are sessile and vulnerable to environmental changes. Many plant genes are thus under environmental control. We are investigating the gene EARLI1, which responds to several environmental stimuli: aluminum stress, cold temperatures, and long-day photoperiods. We are using this gene as a model for identifying molecular components of cold- and light-responsive gene expression. Analysis of these components will contribute to our understanding of how plants adapt to their environment and will provide basic knowledge for crop improvement.
Publications
- Bubier J and Schlappi M (2004) Cold induction of EARLI1, a putative Arabidopsis lipid transfer protein, is light- and calcium-dependent. Plant Cell & Environment (in press).
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Progress 10/01/01 to 09/30/02
Outputs
During the first year of this granting period, our main objective was to determine whether the environmentally regulated gene EARLI1 plays a role in either flowering time and/or cold acclimation of Arabidopsis. EARLI1 is stably activated after vernalization, which promotes flowering in FLC-containing, late-flowering lines of Arabidopsis. We thus hypothesized that EARLI1 may be repressed by FLC. Our new results indicate, however, that EARLI1's response to vernalization (and the other stimuli) is independent of FLC. This result does not confirm the above hypothesis, but suggests that the vernalization response of EARLI1 is FLC-independent. Moreover, overexpression of EARLI1 did not influence flowering time or cold tolerance without a preceding cold acclimation treatment. Loss-of-function analysis of flowering time and cold response effects in plants with a T-DNA insertion in EARLI1 is still in progress, however, preliminary results suggest that the mutation does not
have a significant effect. In order to continue this analysis, we are in the process of knocking out EARLI1 and some or all of its homologs by an RNAi approach. We will particularly focus on the cold acclimation response, because our recent computer modeling of EARLI1 suggests that it is a membrane protein containing a potential motif found in lipid transfer proteins. We also have preliminary data suggesting that a GFP::EARLI1 fusion-protein is localized to the plasma membrane. It is thus conceivable that EARLI1 modifies membranes in response to cold treatment. Our second objective was to identify environment-specific response elements in EARLI1's promoter. Most reporter gene constructs to identify cis-acting elements were finished during the first year of the granting period. The constructs will be introduced into plants and will be analyzed in year 2. This objective will become the major focus for the rest of the granting period, because it will allow us to identify the regulatory
elements for the cold, light, and vernalization responses of EARLI1. These elements together with the identification of interaction proteins will allow us in future experiments to manipulate the environmental regulation of important crop plants.
Impacts
Plants are sessile and vulnerable to environmental changes. Many plant genes are thus under environmental control. We are investigating EARLI1, which responds to several environmental stimuli: aluminum stress, cold shock, long-day photoperiod, and vernalization. We are using this gene as a model for identifying molecular components of environmental regulation. Analysis of these components will contribute to our understanding of how plants interact with their environment and will provide basic knowledge for crop improvement.
Publications
- Bubier, J., Schlappi, M., and Wilkosz, R. (2002) Function and regulation of the vernalization-responsive gene EARLI1. In: Plant Biology2002, 351 (p.96).
- Schlappi, M. and Bubier, J. (2002) Function and regulation of the vernalization-responsive gene EARLI1. In: 13th International Conference on Arabidopsis Research, 8-28.
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