The impact of Arabidopsis thaliana SNF 1‐related‐kinase 1 (Sn RK 1)‐activating kinase 1 (Sn AK 1) and Sn AK 2 on Sn RK 1 phosphorylation status: characterization of a Sn AK double mutant

Summary Arabidopsis thaliana SNF 1‐related‐kinase 1 (Sn RK 1)‐activating kinase 1 (AtSn AK 1) and AtSn AK 2 have been shown to phosphorylate in vitro and activate the energy signalling integrator, Sn RK 1. To clarify this signalling cascade in planta , a genetic‐ and molecular‐based approach was developed. Homozygous single AtSn AK 1 and AtSn AK 2 T‐ DNA insertional mutants did not display an apparent phenotype. Crossing of the single 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 in vitro , were shown to be AtSn AK 1 AtSn AK 2 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 Sn RK 1 catalytic subunit to be non‐phosphorylated in the absence of both Sn AK s. When the double mutant was complemented with a DNA construct containing an AtSn AK 2 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 Sn AK in vivo , and this is correlated with Sn RK 1 phosphorylation. These data show that both Sn AK s are kinases phosphorylating Sn RK 1, and thereby they contribute to energy signalling in planta .