The impact of Arabidopsis thaliana SNF1‐related‐kinase 1 (SnRK1)‐activating kinase 1 (SnAK1) and SnAK2 on SnRK1 phosphorylation status: characterization of a SnAK double mutant

Abstract

Arabidopsis thaliana SNF1-related-kinase 1 (SnRK1)-activating kinase 1 (AtSnAK1) and AtSnAK2 have been shown to phosphorylate in vitro and activate the energy signalling integrator, SnRK1. To clarify this signalling cascade in planta, 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 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 AtSnAK1 AtSnAK2 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 in vivo, and this is correlated with SnRK1 phosphorylation. These data show that both SnAKs are kinases phosphorylating SnRK1, and thereby they contribute to energy signalling in planta.

Dehydration‐responsive nuclear proteome landscape of chickpea (Cicer arietinum L.) reveals phosphorylation‐mediated regulation of stress response

“…Also, to circumvent the restrictions imposed by fixed number of genomic sequences, an array of well‐designed cotranslational and posttranslational layers of regulations and modifications are thus implemented (Guerra et al, ; Natan, Wells, Teichmann, & Marsh, ; Schütze, Harter, & Chaban, ; Withers & Dong, ; Zoschke, Chotewutmontri, & Barkan, ). Phosphorylation, the most abundant posttranslational modifications (PTMs), intimately involved in almost every signalling cascades act as precise molecular triggers (Chen et al, ; Coursol et al, ; Glab et al, ). It also creates exponential increase in the number of mature functional forms of a protein, generated from fixed subset of translational products (Prabakaran et al, ).…”

Upstream kinases of plant SnRKs are involved in salt stress tolerance

“…On 3% sorbitol plates, in which the osmolarity was identical to that of the glucose‐ and sucrose‐supplemented plates, we observed no difference in growth between the wild‐type and the double mutant (Figure S1c). These results indicate that GRIK1 and GRIK2 specifically function in high‐glucose conditions, which is consistent with the reported phenotype of the grik1‐1 grik2‐1 mutant (Glab et al ., ).…”

“…Recently, Glab et al . () have shown that the grik1‐1 grik2‐1 mutant could be rescued by sugar supplementation, yet the mature plant was stunted and sterile. The grik1‐2 grik2‐1 double mutant used here grew and reproduced normally under our growth‐room conditions (Figure c).…”

“…In this study, we successfully produced a grik1‐2 grik2‐1 double mutant with near wild‐type vegetative growth. Previous data had indicated that the grik1‐1 grik2‐1 double mutant was lethal before germination (Bolle et al ., ) or needed supplementation of sugar to grow beyond the cotyledon‐stage (Glab et al ., ). Whereas the grik2‐1 mutant line used here (Salk_015230) is identical to that of previous reports, the mutant lines harbouring T‐DNA insertions in GRIK1 were different.…”

“…The remaining phosphorylation of SnRK1s may be also sufficient to maintain SnRK1‐mediated pathways, as the grik1‐2 grik2‐1 double mutant did not exhibit any defects under dark or submergence conditions (Figure S1a and b), in which SnRK1s play important roles (Baena‐González et al ., ; Cho et al ., ). On the other hand, the grik1‐2 grik2‐1 double mutant was sensitive to high glucose (Figure S1c) as previously shown for the grik1‐1 grik2‐1 double mutant (Glab et al ., ), indicating that the truncated protein encoded by the grik1‐2 allele cannot fully replace the intact GRIK1. Lack of GRIKs might have multiple effects on SnRK1‐mediated pathways, including a decrease in the amount of SnRK1.1 in the grik1‐2 grik2‐1 double mutant (Figure f).…”