Browsing by Author "Martinho, Cláudia"
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- Dissection of miRNA Pathways Using Arabidopsis Mesophyll ProtoplastsPublication . Martinho, Cláudia; Confraria, Ana; Elias, Carlos Alexandre; Crozet, Pierre; Rubio-Somoza, Ignacio; Weigel, Detlef; Baena-González, ElenaMicroRNAs (miRNAs) control gene expression mostly post-transcriptionally by guiding transcript cleavage and/or translational repression of complementary mRNA targets, thereby regulating developmental processes and stress responses. Despite the remarkable expansion of the field, the mechanisms underlying miRNA activity are not fully understood. In this article, we describe a transient expression system in Arabidopsis mesophyll protoplasts, which is highly amenable for the dissection of miRNA pathways. We show that by transiently overexpressing primary miRNAs and target mimics, we can manipulate miRNA levels and consequently impact on their targets. Furthermore, we developed a set of luciferase-based sensors for quantifying miRNA activity that respond specifically to both endogenous and overexpressed miRNAs and target mimics. We demonstrate that these miRNA sensors can be used to test the impact of putative components of the miRNA pathway on miRNA activity, as well as the impact of specific mutations, by either overexpression or the use of protoplasts from the corresponding mutants. We further show that our miRNA sensors can be used for investigating the effect of chemicals on miRNA activity. Our cell-based transient expression system is fast and easy to set up, and generates quantitative results, being a powerful tool for assaying miRNA activity in vivo.
- Mechanisms of regulation of SNF1/AMPK/SnRK1 protein kinasesPublication . Crozet, Pierre; Margalha, Leonor; Confraria, Ana; Rodrigues, Américo; Martinho, Cláudia; Adamo, Mattia; Elias, Carlos A.; Baena-González, ElenaThe SNF1 (sucrose non-fermenting 1)-related protein kinases 1 (SnRKs1) are the plant orthologs of the budding yeast SNF1 and mammalian AMPK (AMP-activated protein kinase). These evolutionarily conserved kinases are metabolic sensors that undergo activation in response to declining energy levels. Upon activation, SNF1/AMPK/SnRK1 kinases trigger a vast transcriptional and metabolic reprograming that restores energy homeostasis and promotes tolerance to adverse conditions, partly through an induction of catabolic processes and a general repression of anabolism. These kinases typically function as a heterotrimeric complex composed of two regulatory subunits, β and γ, and an α-catalytic subunit, which requires phosphorylation of a conserved activation loop residue for activity. Additionally, SNF1/AMPK/SnRK1 kinases are controlled by multiple mechanisms that have an impact on kinase activity, stability, and/or subcellular localization. Here we will review current knowledge on the regulation of SNF1/AMPK/SnRK1 by upstream components, post-translational modifications, various metabolites, hormones, and others, in an attempt to highlight both the commonalities of these essential eukaryotic kinases and the divergences that have evolved to cope with the particularities of each one of these systems.
- miRNAs mediate SnRK1-dependent energy signaling in ArabidopsisPublication . Confraria, Ana; Martinho, Cláudia; Elias, Alexandre; Rubio-Somoza, Ignacio; Baena-González, ElenaThe SnRK1 protein kinase, the plant ortholog of mammalian AMPK and yeast Snf1, is activated by the energy depletion caused by adverse environmental conditions. Upon activation, SnRK1 triggers extensive transcriptional changes to restore homeostasis and promote stress tolerance and survival partly through the inhibition of anabolism and the activation of catabolism. Despite the identification of a few bZIP transcription factors as downstream effectors, the mechanisms underlying gene regulation, and in particular gene repression by SnRK1, remain mostly unknown. microRNAs (miRNAs) are 20-24 nt RNAs that regulate gene expression post-transcriptionally by driving the cleavage and/or translation attenuation of complementary mRNA targets. In addition to their role in plant development, mounting evidence implicates miRNAs in the response to environmental stress. Given the involvement of miRNAs in stress responses and the fact that some of the SnRK1-regulated genes are miRNA targets, we postulated that miRNAs drive part of the transcriptional reprogramming triggered by SnRK1. By comparing the transcriptional response to energy deprivation between WT and dcl1-9, a mutant deficient in miRNA biogenesis, we identified 831 starvation genes misregulated in the dcl1-9 mutant, out of which 155 are validated or predicted miRNA targets. Functional clustering analysis revealed that the main cellular processes potentially co-regulated by SnRK1 and miRNAs are translation and organelle function and uncover TCP transcription factors as one of the most highly enriched functional clusters. TCP repression during energy deprivation was impaired in miR319 knockdown (MIM319) plants, demonstrating the involvement of miR319 in the stress-dependent regulation of TCPs. Altogether, our data indicates that miRNAs are components of the SnRK1 signaling cascade contributing to the regulation of specific mRNA targets and possibly tuning down particular cellular processes during the stress response.