Progress 09/01/05 to 08/31/06
Outputs
Aim 1. To determine the basis for the phenotypic changes observed in arr mutants. Continued characterization of arr mutants has revealed cytokinin-related roles for the type-B ARRs in vascular development and meristem function, as well as a potential role for the type-B ARRs in ethylene signaling. We found that the primary root of an arr1,10,12 mutant prematurely terminates growth and forms only protoxylem, in contrast to wild type roots which make protoxylem and metaxylem. By use of a labile cyclin-GUS fusion protein, we demonstrated that cytokinin treatment reduces the number of cells in the root meristem of wildtype plants undergoing cell division, but this effect is not observed in an arr1,12 double mutant. Evidence that the type-B ARRs are involved in cell division at the shoot meristem was obtained by analysis of an arr1,10,12 mutant. The rosette leaves of the arr1,10,12 plant are smaller than wild type, but the cell size is similar to wild type, indicating that
the leaves are smaller due to a reduction in cell number. In collaboration with Brad Binder (Univ. of Wisconsin), we examined the short-term response of type-B response regulator mutants to ethylene. We find that some arr mutants have a normal growth response to ethylene, but are significantly slower than wt in recovery after ethylene is removed. This suggests that the type-B response regulators may be multi-functional and able to coordinate/participate in several independent signaling pathways. Aim 2. Structure/function analysis of ARRs. We have confirmed that a 35S::ARR1 construct can rescue an arr1,10,12 mutant. To determine the role of phosphorylation in mediating the signal, we have made site-directed mutations of ARR1 and ARR12 so that they no longer contain a phosphorylatable Asp. We have transformed these constructs and controls into wild type, arr1,12, arr1,10,12, and arr1,10,12null to test rescue of cytokinin phenotypes and arr2,10,12 to test rescue of the ethylene
phenotype. To determine if only subfamily-1 type-B ARRs are functionally able to transmit the cytokinin signal we have made constructs in which ARR1 and ARR12 (subfamily 1), ARR20 (subfamily 3), and ARR21 (subfamily 2) are driven by the ARR1 promoter. These constructs have been transformed into wild type and arr1,12 plants. Aim 3. To determine expression changes that take place as a result of type-B ARR action. We are currently examining the transcriptional response of several arr mutants to both cytokinin and ethylene, using both a targeted gene approach as well as microarray analysis.
Impacts
Our results support a general role for the type-B response regulators in mediating the cytokinin transcriptional response in plants. Specific roles have also begun to be determined by examining different mutant combinations. Our findings serve to define how the type-B response regulators control transcription by themselves and in concert with additional transcription factors to regulate the cytokinin response in plants. Our findings also indicate that the type-B response regulators may be multi-functional and able to participate in the ethylene as well as the cytokinin signaling pathway.
Publications
- Chen, Y.-F., Etheridge, N., and Schaller, G.E. (2005) Ethylene signal transduction. Annals Botany 95, 901-915
- Mason, M. G., and Schaller, G.E. (2005) Histidine kinase activity and the regulation of ethylene signal transduction. Can. J. Botany 83, 563-570
- Roshotte, A.M. Mason, M.G., Hutchison, C.E., Ferreira, F.J., Schaller, G.E., and Kieber, J.J. (2006) A subset of Arabidopsis AP2 transcription factors mediates cytokinin responses in concert with a two-component pathway. Proc. Natl. Acad. Sci. USA 103: 11081-11085
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Progress 09/01/04 to 08/31/05
Outputs
Aim 1. To determine the basis for the phenotypic changes observed in arr mutants. We have generated higher order mutant combinations involving new arr2 and arr10 alleles that are predicted to be complete nulls rather than hypomorphic. The new arr1,10,12 triple mutant has reduced stature, premature termination of primary root growth, and is light sensitive. We were unable to generate a double homozygote of arr1,2 with the new arr2 allele, indicating effects of these mutations upon fertility; progeny ratios obtained from crosses indicate that the arr1 and arr2 mutations affect both the male and the female gametophyte. To examine meristem activity, we crossed a wt line containing cyclin-GUS to an arr1,10,12 triple mutant. From this cross we should isolate the triple mutant (whose primary root stops growing and is thus predicted to have reduced meristematic activity) and an arr1,12 mutant (whose primary root is predicted to have increased meristematic acitivity). Aim 2.
Structure/function analysis of ARRs. We are constructing site-directed mutatations in ARR12 at the phosphorylatable Asp as a means to test the role of phosphorylation in mediating the cytokinin response. We are also making constructs that use the promoter of ARR1 to drive expression of other type-B ARRs, including a member each of subfamily 1, 2, and 3. Aim 3. To determine expression changes that take place as a result of type-B ARR action. We are using microarray analysis to examine how the arr mutations affect the cytokinin response. Significant reductions in cytokinin-regulated gene expression are seen in an arr1,12 double mutant compared to wildtype. Cytokinin-regulated gene expression is further reduced in an arr1,10,12 triple mutant. These data support a central role for the type-B ARRs in regulating the primary transcriptional response of plants to cytokinin. Of interest is the finding that a group of genes induced by cytokinin in wildtype plants, is repressed by cytokinin in
the arr1,12 mutant. This result suggests that that the type-B ARRs may act as both activators and repressors of transcription for the same gene set, with their transcriptional role dependent on additional factors present in their local cellular environment. In collaboration with Dr. Joe Kieber (UNC-Chapel Hill), we have used microarray analysis to determine the expression changes mediated by another set of cytokinin-regulated transcription factors (the CRF family). Triple crf mutants show significant reductions in cytokinin-regulated gene expression. Of particular interest is our finding that the genes affected by the triple crf mutant substantially overlap with the gene set regulated by the type-B ARRs. Thus, both ARRs and CRFs function in the coordinate regulation of the primary transcriptional response to cytokinin.
Impacts
Our results support a general role for the type-B response regulators in mediating the cytokinin transcriptional response in plants. Specific roles have also begun to be determined by examining different mutant combinations. Our findings serve to define how the type-B response regulators control transcription by themselves and in concert with additional transcription factors to regulate the cytokinin response in plants.
Publications
- Mason, M.G., Mathews, D.M., Argyros D.A., Maxwell B.B., Kieber J.J., Alonso J.M., Ecker J.R., Schaller G.E. (2005) Multiple type-B response regulators mediate cytokinin signal transduction in Arabidopsis. Plant Cell. 17, 3007-3018.
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