Involvement of the eIF2α Kinase GCN2 in UV-B Responses.

Paula Llabata,Jose Gadea,Karolina Słomiňska-Durdasiak,Lukas Hubert Zeh,Isabel Faus,Marie-Theres Hauser,Julia Richter

doi:10.3389/fpls.2019.01492

Abstract

GCN2 (general control nonrepressed 2) is a serine/threonine-protein kinase that regulates translation in response to stressors such as amino acid and purin deprivation, cold shock, wounding, cadmium, and UV-C exposure. Activated GCN2 phosphorylates the α-subunit of the eukaryotic initiation factor 2 (eIF2) leading to a drastic inhibition of protein synthesis and shifting translation to specific mRNAs. To investigate the role of GCN2 in responses to UV-B radiation its activity was analyzed through eIF2α phosphorylation assays in mutants of the specific UV-B and stress signaling pathways of Arabidopsis thaliana. EIF2α phosphorylation was detectable 30 min after UV-B exposure, independent of the UV-B photoreceptor UV RESISTANCE LOCUS8 and its downstream signaling components. GCN2 dependent phosphorylation of eIF2α was also detectable in mutants of the stress related MAP kinases, MPK3 and MPK6 and their negative regulator map kinase phosphatase1 (MKP1). Transcription of downstream components of the UV-B signaling pathway, the Chalcone synthase (CHS) was constitutively higher in gcn2-1 compared to wildtype and further increased upon UV-B while GLUTATHIONE PEROXIDASE7 (GPX7) behaved similarly to wildtype. The UVR8 independent FAD-LINKED OXIDOREDUCTASE (FADox) had a lower basal expression in gcn2-1 which was increased upon UV-B. Since high fluence rates of UV-B induce DNA damage the expression of the RAS ASSOCIATED WITH DIABETES PROTEIN51 (RAD51) was quantified before and after UV-B. While the basal expression was similar to wildtype it was significantly less induced upon UV-B in the gcn2-1 mutant. This expression pattern correlates with the finding that gcn2 mutants develop less cyclobutane pyrimidine dimers after UV-B exposure. Quantification of translation with the puromycination assay revealed that gcn2 mutants have an increased rate of translation which was also higher upon UV-B. Growth of gcn2 mutants to chronic UV-B exposure supports GCN2’s role as a negative regulator of UV-B responses. The elevated resistance of gcn2 mutants towards repeated UV-B exposure points to a critical role of GCN2 in the regulation of translation upon UV-B.

Highlights

UV-B (280–315 nm) is the most harmful radiation of the sun’s spectrum reaching the biosphere
To investigate the role of GCN2 in responses to UV-B its activity was analyzed through eIF2α phosphorylation assays in wildtype and gcn2-1 mutants
We determined whether cellulose diacetate filtered UV-B stimulates eIF2α phosphorylation and when eIF2α phosphorylation is detectable after the start of the UV-B exposure (Figures 1B, C)

Summary

Introduction

UV-B (280–315 nm) is the most harmful radiation of the sun’s spectrum reaching the biosphere. Monomeric UVR8 interacts with a key regulator of photomorphogenesis, the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). The UVR8/COP1 interaction is crucial for the expression and stability of the transcription factors ELONGATED HYPOCOTYL5 (HY5) and its homolog HYH (Ulm et al, 2004; Stracke et al, 2010; Rizzini et al, 2011; Huang et al, 2013; Binkert et al, 2014). Brown and Jenkins (2008) found that UVR8 dependent and independent genes exhibit different needs for fluence rates. Among low fluence rate UVR8 dependent genes are HY5, HYH, and their downstream targets CHALCONE SYNTHASE (CHS) and GLUTATHIONE PEROXIDASE7 (GPX7). Among the UV-B induced but UVR8 independent genes are for example FAD-LINKED OXIDOREDUCTASE (FADox) (Brown and Jenkins, 2008)

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