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Basic study of cell cycle genes

Heberle-Bors' group has studied a MAPkinase pathway that is involved in cytokinesis. They identified this pathway first in tobacco and are now also isolating the corresponding genes from Arabidopsis. The following genes were isolated:
- NtMEK1: a tobacco MAP kinase, which activates the tobacco MAP kinase Ntf6,
- ATMPK16: a MAP kinase, which is the putative Arabidopsis homologue of Ntf6,
- AtMKK6: a MAP kinase, which is the putative Arabidopsis homologue of NtMEK1.

A transposon insertion line was found for ATMPK16. Screening of selfed plants has been done to identify homozygous lines. Furthermore, GFP-ATMPK16 transgenic lines were produced for visualization of MAPK localization to the phragmoplast. Three GFP-alone transgenics showed GFP expression. Four GFP-ATMPK16 transgenics only show fluorescence at the leaf tip (not seen in WT). GFP-ATMPK16 fusion is functional in yeast assays. The tobacco homologe Ntf6 was also cloned as a GFP fusion (fluorescent after particle bombardment of pollen), the NtMEK1 as a RFP fusion, so that co-localisation of Ntf6 and NtMEK1 can be observed.

A Guanine nucleotide dissociation inhibitor (GDI) was shown to physically interact with Ntf6 and to alter kinase activity of Ntf6 and NtMEK1. This raises the possibility that GDI is an Ntf6 substrate (first substrate of a plant MAPK demonstrated). The Arabidopsis MAP kinase ATMPK13 is the putative orthologue of the tobacco MAP kinase Ntf6. This latter kinase has been implicated in the process of cytokinesis in tobacco cells. Attempts were therefore made to elucidate the role of ATMPK13 in Arabidopsis.

A transgenic plant line with a transposon insertion in the ATMPK13 gene was identified. Although this line displayed an interesting phenotype (viz. the absence of petals), it was shown genetically that this was not due to the transposon insertion in the ATMPK13 gene, and was probably due to an insertion in another gene. Indeed, Southern analysis showed the presence of at least 6 transposons in this particular line. Because of the difficulty of purifying the ATMPK13 insertion from the other transposons, together with the mobile nature of the transposon, work on this line was discontinued. A T-DNA insertion line is presently being analysed.

In an attempt to identify the subcellular localization of ATMPK13, a GFP-ATMPK13 fusion was constructed. GFP fluorescence of the fusion protein and the functionality of the kinase as a fusion protein were demonstrated in yeast using functional assays. Four transgenic GFP-ATMPK13 transformants were obtained, but none of them showed GFP fluorescence (the control lines with GFP alone were strongly fluorescent).

A peptide antibody was raised against the N-terminus of ATMPK13, but the quality of the antibody was poor. In Western analysis it recognised a band only in flowers. By contrast an antibody against the C-terminus (provided by L. Bogre) recognised a band in flowers, roots, stems, and seedlings. The reason for this discrepancy is unclear. (Qualitative) RT-PCR demonstrated the presence of an ATMPK13 transcript in all tissues except cauline leaves and siliques. The MAP kinase MEK1 is an activator of Ntf6, and has been shown to function in cytokinesis. The putative Arabidopsis orthologue of MEK1 is MKK6. An MKK6 cDNA was isolated. Studies in yeast showed that MKK6 can activate ATMPK13 - a yeast strain mutated in the MPK1 MAP kinase is only complemented when MKK6 and ATMPK13 are co-expressed, and higher kinase activity can be immunoprecipitated from the double transforming.

Therefore the MKK6/ATMPK13 pair of kinases show similar behaviour to the tobacco MEK1/Ntf6 pair. Indeed MEK1 can activate ATMPK13 in yeast functional assays. While it has been possible to show that ATMPK13 encodes a functional kinase molecule in yeast, it has proved to be recalcitrant to studies in bacteria and after re-introduction into the plant. Possibly post-translation modifications and/or a particular sub-cellular localization are required for proper ATMPK13 function.

Reported by

University of Vienna
Institute of Microbiology and Genetics, Dr Bohrgasse 9
1030 Wien
Austria
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