The impact of Arabidopsis thaliana SNF1-related-kinase1 (SnRK1)-activating kinase1 (SnAK1) and SnAK2 on SnRK1 phosphorylation status: characterization of a SnAK double mutant.

Abstract

Arabidopsis thaliana SNF1-related-kinase1 (SnRK1)-activating kinase1 (AtSnAK1) and AtSnAK2 have been shown to phosphorylate invitro and activate the energy signalling integrator, SnRK1. To clarify this signalling cascade inplanta, a genetic- and molecular-based approach was developed. Homozygous single AtSnAK1 and AtSnAK2 T-DNA insertional mutants did not display an apparent phenotype. Crossing of thesingle mutants did not allow the isolation of double-mutant plants, whereas self-pollinating the S1-/-S2+/- sesquimutant specifically gave approximatively 22% individuals in their offspring that, when rescued on sugar-supplemented media invitro, were shown to be AtSnAK1AtSnAK2 double mutants. Interestingly, this was not obtained in the case of the other sesquimutant, S1+/-S2-/-. Although reduced in size, the double mutant had the capacity to produce flowers, but not seeds. Immunological characterization established the T-loop of the SnRK1 catalytic subunit to be non-phosphorylated in the absence of both SnAKs. When the double mutant was complemented with a DNA construct containing an AtSnAK2 open reading frame driven by its own promoter, a normal phenotype was restored. Therefore, wild-type plant growth and development is dependent on the presence of SnAK invivo, and this is correlated with SnRK1 phosphorylation. These data show that both SnAKs are kinases phosphorylating SnRK1, and thereby they contribute to energy signalling inplanta.