Publication details

Investigating the role of sensor histidine kinase CKI1 in Arabidopsis using a reverse genetics approach.



Year of publication 2003
Type Article in Proceedings
Conference Book of Abstracts, Plant GEMs/GARNet, Plant Genomic European Meetings, York, U.K.
MU Faculty or unit

Faculty of Science

Field Genetics and molecular biology
Keywords female gametophyte development; two-component signaling; sensor histidine kinase; early seed development; genomic imprinting
Description The CKI1 gene was isolated using activation mutagenesis by Tatsuo Kakimoto (1996, Science 274: 982-985). Based on the phenotype of Arabidopsis calli ectopically over-expressing CKI1 and the significant degree of similarity with bacterial two-component sensor histidine kinases, CKI1 was implicated in cytokinin sensing in Arabidopsis. However, in spite of various experimental efforts, neither the genuine ligand, nor the biological function of the gene was identified. We used reverse genetics approach employing unstable En-1 element to elucidate the role of CKI1 in Arabidopsis development. We isolated a loss-of-function mutation in CKI1 resulting from an insertion of En-1 transposon into the CKI1 coding sequence. No plants homozygous for the cki1-i were found in the progeny of CKI1/cki1-i lines. The subsequent genetic analysis revealed that mutant cki1-i allele could not be transmitted through the female germ line. Confocal laser scanning microscopy identified a block in megagametogenesis characterized by the abortion of the central vacuole in mutant embryo sacs and degradation of the developing female gametophyte after completion of all mitotic divisions. Two independent stable mutant alleles and one revertant wild type allele resulting from En-1 excision confirmed unambiguously a causal link between the cki1-i and the phenotype. In situ localization of CKI1 mRNA and histochemical analysis of stable transformants harboring the uidA gene under the control of CKI1 promoter confirmed the expression of CKI1 in a female gametophyte tissue. The CKI1 expression pattern indicates that the chain of molecular and subcellular events resulting in the phenotype manifestation can be triggered very early in megagametogenesis. The expression of paternally transmitted CKI1 early after fertilization was identified, which might imply a role for CKI1 in endosperm development. This may also suggest that the concept of genome-wide male imprinting during early seed development needs to be reconsidered. The results provide the first evidence that gametophytic expression of a sensor-like molecule is essential for specific processes during megagametogenesis and demonstrate the power of reverse genetics approach using unstable En-1 element.
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