ABI1 and PP2CA Phosphatases Are Negative Regulators of Snf1-Related Protein Kinase1 Signaling in Arabidopsis

Plant survival under environmental stress requires the integration of multiple signaling pathways into a coordinated response, but the molecular mechanisms underlying this integration are poorly understood. Stress-derived energy deprivation activates the Snf1-related protein kinases1 (SnRK1s), triggering a vast transcriptional and metabolic reprogramming that restores homeostasis and promotes tolerance to adverse conditions. Here, we show that two clade A type 2C protein phosphatases (PP2Cs), established repressors of the abscisic acid (ABA) hormonal pathway, interact with the SnRK1 catalytic subunit causing its dephosphorylation and inactivation. Accordingly, SnRK1 repression is abrogated in double and quadruple pp2c knockout mutants, provoking, similarly to SnRK1 overexpression, sugar hypersensitivity during early seedling development. Reporter gene assays and SnRK1 target gene expression analyses further demonstrate that PP2C inhibition by ABA results in SnRK1 activation, promoting SnRK1 signaling during stress and once the energy deficit subsides. Consistent with this, SnRK1 and ABA induce largely overlapping transcriptional responses. Hence, the PP2C hub allows the coordinated activation of ABA and energy signaling, strengthening the stress response through the cooperation of two key and complementary pathways.

SUMOylation represses SnRK1 signaling in Arabidopsis

The SnRK1 protein kinase balances cellular energy levels in accordance with extracellular conditions and is thereby key for plant stress tolerance. In addition, SnRK1 has been implicated in numerous growth and developmental processes from seed filling and maturation to flowering and senescence. Despite its importance, the mechanisms that regulate SnRK1 activity are poorly understood. Here, we demonstrate that the SnRK1 complex is SUMOylated on multiple subunits and identify SIZ1 as the E3 Small Ubiquitin-like Modifier (SUMO) ligase responsible for this modification. We further show that SnRK1 is ubiquitinated in a SIZ1-dependent manner, causing its degradation through the proteasome. In consequence, SnRK1 degradation is deficient in siz1-2 mutants, leading to its accumulation and hyperactivation of SnRK1 signaling. Finally, SnRK1 degradation is strictly dependent on its activity, as inactive SnRK1 variants are aberrantly stable but recover normal degradation when expressed as SUMO mimetics. Altogether, our data suggest that active SnRK1 triggers its own SUMOylation and degradation, establishing a negative feedback loop that attenuates SnRK1 signaling and prevents detrimental hyperactivation of stress responses.

Concerted involvement of Cdx/Hox genes and Wnt signaling in morphogenesis of the caudal neural tube and cloacal derivatives from the posterior growth zone

Decrease in Cdx dosage in an allelic series of mouse Cdx mutants leads to progressively more severe posterior vertebral defects. These defects are corrected by posterior gain of function of the Wnt effector Lef1. Precocious expression of Hox paralogous 13 genes also induces vertebral axis truncation by antagonizing Cdx function. We report here that the phenotypic similarity also applies to patterning of the caudal neural tube and uro-rectal tracts in Cdx and Wnt3a mutants, and in embryos precociously expressing Hox13 genes. Cdx2 inactivation after placentation leads to posterior defects, including incomplete uro-rectal septation. Compound mutants carrying one active Cdx2 allele in the Cdx4-null background (Cdx2/4), transgenic embryos precociously expressing Hox13 genes and a novel Wnt3a hypomorph mutant all manifest a comparable phenotype with similar uro-rectal defects. Phenotype and transcriptome analysis in early Cdx mutants, genetic rescue experiments and gene expression studies lead us to propose that Cdx transcription factors act via Wnt signaling during the laying down of uro-rectal mesoderm, and that they are operative in an early phase of these events, at the site of tissue progenitors in the posterior growth zone of the embryo. Cdx and Wnt mutations and premature Hox13 expression also cause similar neural dysmorphology, including ectopic neural structures that sometimes lead to neural tube splitting at caudal axial levels. These findings involve the Cdx genes, canonical Wnt signaling and the temporal control of posterior Hox gene expression in posterior morphogenesis in the different embryonic germ layers. They shed a new light on the etiology of the caudal dysplasia or caudal regression range of human congenital defects.

Compartment-dependent activities of Wnt3a/β-catenin signaling during vertebrate axial extension

Extension of the vertebrate body results from the concerted activity of many signals in the posterior embryonic end. Among them, Wnt3a has been shown to play relevant roles in the regulation of axial progenitor activity, mesoderm formation and somitogenesis. However, its impact on axial growth remains to be fully understood. Using a transgenic approach in the mouse, we found that the effect of Wnt3a signaling varies depending on the target tissue. High levels of Wnt3a in the epiblast prevented formation of neural tissues, but did not impair axial progenitors from producing different mesodermal lineages. These mesodermal tissues maintained a remarkable degree of organization, even within a severely malformed embryo. However, from the cells that failed to take a neural fate, only those that left the epithelial layer of the epiblast activated a mesodermal program. The remaining tissue accumulated as a folded epithelium that kept some epiblast-like characteristics. Together with previously published observations, our results suggest a dose-dependent role for Wnt3a in regulating the balance between renewal and selection of differentiation fates of axial progenitors in the epiblast. In the paraxial mesoderm, appropriate regulation of Wnt/β-catenin signaling was required not only for somitogenesis, but also for providing proper anterior-posterior polarity to the somites. Both processes seem to rely on mechanisms with different requirements for feedback modulation of Wnt/β-catenin signaling, once segmentation occurred in the presence of high levels of Wnt3a in the presomitic mesoderm, but not after permanent expression of a constitutively active form of β-catenin. Together, our findings suggest that Wnt3a/β-catenin signaling plays sequential roles during posterior extension, which are strongly dependent on the target tissue. This provides an additional example of how much the functional output of signaling systems depends on the competence of the responding cells.