Are histidine kinases involved in cytokinin signal transduction? Application of a gene knock-out based function search strategy.
| Authors | |
|---|---|
| Year of publication | 1998 |
| Type | Article in Proceedings |
| Conference | 8th Days of Plant Physiology; Book of abstracts |
| MU Faculty or unit | |
| Citation | |
| Field | Genetics and molecular biology |
| Keywords | cytokinin receptor; En-1 insertion mutagenesis; PCR-based screen |
| Description | Receptors showing histidine kinase activity coupled with signal transduction proteins (response regulators) and constituting so called two-component signaling system, have been well established in bacteria as signal uptake molecules implicated in responses to variety of extracellular stimuli (tab.1, pic.1). Recently, homologs to bacterial histidine kinase receptors and response regulators have been found to be involved in signaling cascades in eukaryotes (tab.2, pic.2). To the well characterised eukaryotical receptor systems with histidine kinase activity belonging above all ethylene uptake in plants (Fluhr 1998) and osmoregulation in yeast (Posas et al. 1996). Using T-DNA activation tagging in Arabidopsis a gene designated CKI-1 (cytokinin independent) has been cloned encoding putative protein with high degree of homology to both prokaryotical and eukaryotical hisidine kinases (Kakimoto 1996, pic.3). Plants transformed with cDNA of CKI-1 gene under CaMV 35S rRNA promotor were able to form green calli and proliferate on medium lacking cytokinin. Therefore, CKI-1 protein has been proposed to be putative cytokinin receptor (Kakimoto 1996). BLAST similarity search (Altschul et al., 1990) in Arabidopsis protein database revealed significant degree of CKI-1 protein similarity to well characterised proteins with histidine kinase activity (Etr1) as well as to putative histidine kinases, products of Arabidopsis sequencing project (Atdb, pic.4). These findings support certain significance of histidine kinases in Arabidopsis thaliana. metabolism. To confirm possible role of CKI-1 in cytokinin perception, we have decided for reverse genetics approach. We screened Arabidopsis thaliana population of about 3000 lines carrying 15.000 independent insertions of the autozomous En-1 maize transposable element (Weissman et al. 1998). Principles of the three-dimensional reverse genetic PCR screen are shown in pic.5. Using this strategy we have identified one positive plant bearing insertion in CKI-1 gene. By the sequencing of PCR product we positioned En-1 element into exon 5 (pic.6). The progeny analysis of the positive screened plant revealed interesting phenotype, but unfortunately because of patent application we can not public our data yet. Exon 5, where the En-1 insertion occur has been predicted to be an extracellular part of putative CKI-1 protein and therefore (concerning physical-chemical properties of proposed ligand) the extracellular loop could be relevant to signal input domain of CKI-1. This hypothesis has been supported by BLAST search homology results (Altschul et al. 1997), where the query has been extracellular part of CKI-1 protein. In the middle of putative extracellular loop we have found significant degree of similarity to nucleotide binding motif of human terminal transferase (Yang et al., 1994, pic.7), suggesting possible interaction of CKI-1 protein with cytokinins, which are purin derivates. These our findings together with Kakimoto results provide strong evidence that CKI-1 could play an important role in Arabidopsis thaliana cytokinin perception. Our further work is aimed to carry out binding assays in order to confirm CKI-1 and cytokinin interaction, making more accurate phenotype analysis of CKI-1 knock-out mutant and to identify other possible members of cytokinin signaling pathway. |
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