Involvement of the eIF2α Kinase GCN2 in UV-B Responses

Abstract

<i>GCN2</i> (<i>general control nonrepressed 2</i>) 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 <i>GCN2</i> 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 <i>GCN2</i> 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 <i>Arabidopsis thaliana</i>. EIF2α phosphorylation was detectable 30 min after UV-B exposure, independent of the UV-B photoreceptor <i>UV RESISTANCE LOCUS8</i> and its downstream signaling components. GCN2 dependent phosphorylation of eIF2α was also detectable in mutants of the stress related MAP kinases, <i>MPK3</i> and <i>MPK6</i> and their negative regulator <i>map kinase phosphatase1</i> (<i>MKP</i>1). Transcription of downstream components of the UV-B signaling pathway, the <i>Chalcone synthase</i> (<i>CHS</i>) was constitutively higher in <i>gcn2-1</i> compared to wildtype and further increased upon UV-B while <i>GLUTATHIONE PEROXIDASE7</i> (<i>GPX7</i>) behaved similarly to wildtype. The UVR8 independent <i>FAD-LINKED OXIDOREDUCTASE</i> (<i>FADox</i>) had a lower basal expression in <i>gcn2-1</i> which was increased upon UV-B. Since high fluence rates of UV-B induce DNA damage the expression of the <i>RAS ASSOCIATED WITH DIABETES PROTEIN51</i> (<i>RAD51</i>) 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 <i>gcn2-1</i> mutant. This expression pattern correlates with the finding that <i>gcn2</i> mutants develop less cyclobutane pyrimidine dimers after UV-B exposure. Quantification of translation with the puromycination assay revealed that <i>gcn2</i> mutants have an increased rate of translation which was also higher upon UV-B. Growth of <i>gcn2</i> mutants to chronic UV-B exposure supports <i>GCN2</i>'s role as a negative regulator of UV-B responses. The elevated resistance of <i>gcn2</i> mutants towards repeated UV-B exposure points to a critical role of <i>GCN2</i> in the regulation of translation upon UV-B.