Rolland, F. | bioRxiv Science

bioRxiv2023

S-acylation and tonoplast localization of the Geminivirus Rep-Interacting Kinase/SnRK1-Activating Kinase (GRIK/SnAK) proteins differentially regulate salt and energy stress responses in Arabidopsis

SnRK1 and SnRK3.11/SOS2 are key protein kinases in plant cellular energy and salt stress signaling, respectively. Using cellular assays, we confirm that the GRIK/SnAK (Geminivirus Rep-Interacting Kinase/SnRK1-Activating Kinase) proteins act as their main activating upstream kinases in Arabidopsis, catalyzing T-loop phosphorylation on the SnRK11 T175 and SOS2 T168 residues. Remarkably, SnRK11 phosphorylation on the neighbouring S176 residue competes with GRIK-mediated T175 phosphorylation to negatively regulate SnRK1 activity. Cellular assays and transgenic plants also revealed that the GRIK proteins, via N-terminal S-acylation, are predominantly localized at the tonoplast, where they interact with SnRK11 and SOS2. We optimized a leaf mesophyll protoplast-based Acyl PEG Exchange (APE) protocol to further explore GRIK protein S-acylation and tonoplast recruitment and identified the amino acid residues involved. GRIK1 tonoplast localization is likely mediated by initial membrane sampling via N-terminal domain hydrophobicity and local S-acylation, independently of a secretory pathway. Finally, grik1-1 grik2-1 double KO mutants complemented with a non-S-acylatable mutant GRIK1 protein exhibit increased salt sensitivity (reduced SOS2 activity) but hyperactive SnRK1 signaling, demonstrating the differential importance of GRIK subcellular localization for Arabidopsis salt and energy stress responses.

plant biology↗

bioRxiv2021

ABA represses TOR and root meristem activity through nuclear exit of the SnRK1 kinase

The phytohormone abscisic acid (ABA) promotes plant tolerance to major stresses like drought, partly by modulating plant growth. However, the underlying mechanisms are poorly understood. Here, we show that cell proliferation in the Arabidopsis thaliana root meristem is controlled by the interplay between three kinases, SNF1-RELATED KINASE 2 (SnRK2), the main driver of ABA signaling, the SnRK1 energy sensor, and the growth-promoting TARGET OF RAPAMYCIN (TOR) kinase. Under favorable conditions, the SnRK11 catalytic subunit is enriched in the nuclei of root cells and this is accompanied by normal cell proliferation and meristem size. Depletion of SnRK2s in a snrk2.2 snrk2.3 double mutant causes constitutive cytoplasmic localization of SnRK11 and a reduction in meristem size, suggesting that, under non-stress conditions, SnRK2s enable growth by retaining SnRK11 in the nucleus. In response to elevated ABA levels, SnRK11 translocates to the cytoplasm and this is accompanied by inhibition of TOR, decreased cell proliferation and meristem size. Blocking nuclear export with leptomycin B abrogates ABA-driven SnRK11 relocalization to the cytoplasm and the inhibition of TOR. Fusion of SnRK11 to an SV40 nuclear localization signal leads to defective TOR repression in response to ABA, demonstrating that SnRK11 nuclear exit is a premise for this repression. Altogether, we demonstrate that SnRK2-dependent changes in SnRK11 subcellular localization are crucial for the regulation of TOR activity and root growth in response to ABA. Such swift relocalization of key regulators may represent a more general strategy of sessile organisms like plants to rapidly respond to environmental changes.

plant biology↗