Progress 09/01/02 to 08/31/04
Outputs
This research focused on the study of the enzymes that are the determining step in cytokinin synthesis, isopentenyl transferases. These enzymes have recently been discovered in the model plant Arabidopsis thaliana as a direct result of the completion of its genome sequencing project(1,2). While all of the objectives have not yet been fulfilled, substantial progress has been made that will result in significant contributions to the field within the next year. The first objective was to identify and develop Arabidopsis plants with reduced levels of isopentenyl transferase transcripts and analyze their impacts on growth and development. Towards this end, insertional mutants from Joseph Ecker's collection at the Salk Institute and Jonathan Clarke's collection at the John Innes Centre have been obtained that disrupt the transcript levels of four of the seven isopentenyl transferase genes. Triple mutants have been generated, and crosses for the quadruple mutant are underway.
The construction of transgenes that disrupt the remaining isopentenyl transferase transcripts by RNA interference has been completed and introduced into one of the triple insertional mutants. Two RNA interference contructs were designed: one is under the control of the strong constitutive promoter CaMV35S, and the second is under the transcriptional control of a glucocorticoid-inducible system (GVG,3). Preliminary analyses of the phenotypes of these plants are currently underway, as are analyses of their transcript abundance. Some of the inducible RNA interference lines placed within the triple insertional mutant show a striking reduction in shoot growth in a glucocorticoid-dependent manner. The second objective was to develop plants that over-produce isopentenyl transferases to study the effects of increased cytokinin production on plant growth and development. Towards that end, both the Agrobacterium tumefaciens isopentenyl transferase gene and one of the Arabidopsis isopentenyl
transferase genes were placed under the control of the GVG system. The resulting transgenic seedlings have small shoots and short roots in a glucocorticoid-dependent manner. However, the transgenic plants harboring the Arabidopsis version of the transgenes have a slightly different phenotype (yellow cotyledons that green after 5 days) when compared to the bacterial gene (green cotyledons), and this will be explored in future work. A mutagenesis screen based on this phenotype is underway. The third objective was to monitor gene expression levels in the plants identified from the first two objectives. Real-time PCR conditions have been worked out for the Arabidopsis isopentenyl transferase transcripts to confirm the degree that these transcripts are altered in the mutants. Whole-genome transcriptional profiling experiments of the overexpression lines are in the planning stages, and will also be performed on selected lines that have reduced isopentenyl transferase transcripts in the
glucocorticoid inducible system. REFERENCES 1.Kakimoto (2001) Plant Cell and Physiology 42(7):677-685 2.Takei et al. (2001) JBC 276:26405-26410 3.Aoyama & Chua (1997) Plant Journal 11(3):605-612
Impacts
Cytokinin is one of the first plant hormones discovered, but until 2001 the enzyme responsible for its synthesis in plants was a mystery. We will capitalize on this recent discovery to uncover how the plant manipulates intrinsic cytokinin levels to coordinate its growth and development. We are generating plants that do not have the ability to make this hormone to discover the processes that absolutely require it, and we are generating plants that make too much of the hormone to discover which processes are sensitive to it. Ultimately these findings will be useful to plant breeders so that they can include the manipulation of this hormone into their plans to shape the overall growth and development of plants to increase crop production.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
The current research focuses on the study of the enzymes that are the determining step in cytokinin synthesis, isopentenyl transferases. These enzymes have recently been discovered in the model plant Arabidopsis thaliana as a direct result of the completion of its genome sequencing project. Substantial progress has been made in the first year of funding toward each objective. The first objective is to identify and develop Arabidopsis plants with reduced levels of isopentenyl transferase transcripts and analyze their impacts on growth and development. Towards this end, insertional mutants from Joseph Ecker's collection at the Salk Institute and Jonathan Clarke's collection at the John Innes Centre have been obtained that disrupt the transcript levels of four of the seven isopentenyl transferase genes. Crosses have been made to generate double mutants, and higher-order crosses are underway. The construction of transgenes that will disrupt the remaining isopentenyl
transferase transcripts by RNA interference is also underway. In the second year the transformation of the quadruple insertional mutant with the RNA interference construct will lead to the generation of plants that lack transcripts for each isopentenyl transferase gene. Physiological studies of the insertional mutants are ongoing. Early results show remarkable redundancy of these enzymes because most processes seem normal. Our most sensitive assay for cytokinin action is a root elongation assay, and only one of the single insertional mutants consistently has roots longer than the controls. Since cytokinins inhibit root growth, the implication of this result is that this mutant has less cytokinin and therefore its root growth is not as inhibited as the control. This assay and others will be performed on the higher-order mutants to determine the physiological impact of removing cytokinins from plant tissues by removing this key enzyme for the hormone's synthesis. The reduction in
cytokinin levels will be confirmed by our collaborators via HPLC-coupled mass spectrophotometry. The second objective was to develop plants that over-produce isopentenyl transferases to study the effects of increased cytokinin production on plant growth and development. Towards that end, both the Agrobacterium tumefaciens isopentenyl transferase gene (the bacterial gene involved in crown gall disease) and one of the Arabidopsis isopentenyl trasferase genes were placed in transgenic constructs so that they would be expressed only when given the testosterone homolog dexamethasone and have antibody tags to detect the levels of their proteins once expressed. Both constructs were introduced into Arabidopsis, and the transgenic plants identified based on co-transformed resistance genes. Analysis of their phenotypes is on-going. The third objective is to monitor gene expression levels in the plants identified from the first two objectives. Real-time PCR conditions have been worked out for
the Arabidopsis isopentenyl transferase transcripts to confirm the degree that these transcripts are altered in the mutants.
Impacts
Cytokinin is one of the first plant hormones discovered, but until 2001 the enzyme responsible for its synthesis in plants was a mystery. We will capitalize on this recent discovery to uncover how the plant manipulates intrinsic cytokinin levels to coordinate its growth and development. We are generating plants that do not have the ability to make this hormone to discover the processes that absolutely require it, and we are generating plants that make too much of the hormone to discover which processes are sensitive to it. Ultimately these findings will be useful to plant breeders so that they can include the manipulation of this hormone into their plans to shape the overall growth and development of plants to increase crop production.
Publications
- No publications reported this period
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