ArabidopsiseIF2α kinase GCN2 is essential for growth in stress conditions and is activated by wounding
- Sébastien Lageix1, 3,
- Elodie Lanet2,
- Marie-Noëlle Pouch-Pélissier1,
- Marie-Claude Espagnol3,
- Christophe Robaglia2,
- Jean-Marc Deragon†1Email author and
- Thierry Pélissier†1
© Lageix et al; licensee BioMed Central Ltd. 2008
Received: 08 September 2008
Accepted: 24 December 2008
Published: 24 December 2008
Phosphorylation of eIF2α provides a key mechanism for down-regulating protein synthesis in response to nutrient starvation or stresses in mammalian and yeast cells. However, this process has not been well characterized in plants
We show here that in response to amino acid and purine starvations, UV, cold shock and wounding, the Arabidopsis GCN2 kinase (AtGCN2) is activated and phosphorylates eIF2α. We show that AtGCN2 is essential for plant growth in stress situations and that its activity results in a strong reduction in global protein synthesis.
Our results suggest that a general amino acid control response is conserved between yeast and plants but that the plant enzyme evolved to fulfill a more general function as an upstream sensor and regulator of diverse stress-response pathways. The activation of AtGCN2 following wounding or exposure to methyl jasmonate, the ethylene precursor 1-Aminocyclopropane-1-carboxylic acid (ACC) and salicylic acid, further suggests that this enzyme could play a role in plant defense against insect herbivores.
Phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) provides a key mechanism for down-regulating protein synthesis in response to nutrient starvation or stresses [1, 2]. Vertebrates use four different eIF2α-kinases (PKR, PERK, HRI and GCN2) to respond to various stresses, and typically one kinase has a predominant role in response to a specific cellular stress condition. For example, in mammals, GCN2 is the primary eIF2α-kinase in response to nutrient limitation , PERK modulates gene expression in response to protein misfolding in the endoplasmic reticulum (ER) , PKR participates in an antiviral pathway mediated by interferon , and HRI couples protein synthesis (predominantly globin) to the availability of heme in the erythroid cell lineage . However, this specialization of function is not complete and, for example, GCN2 is a secondary eIF2α-kinase in response to ER stress in mouse . Arthropods make a more or less specialized use of eIF2α-kinases. The mosquito Anopheles gambiae has three distinct enzymes (HRI, PERK and GCN2) while Drosophila melanogaster and Caenorhabditis elegans have only two (PERK and GCN2) . The situation is similar for fungi since Schizosaccharomyces pombe has three enzymes (two distinct HRI and a GCN2) while Saccharomyces cerevisiae and Neurospora crassa have a single eIF2α-kinase (GCN2) .
In amino acid starved S. cerevisiae cells, GCN2-dependent eIF2α phosphorylation leads to a general down regulation of protein synthesis and to the increased translation of the GCN4 mRNA . Following translation, the GCN4 transcriptional activator can induce the transcription of more than 500 genes, including the majority involved in amino acid synthesis. Uncharged tRNAs that accumulate during amino acid starvation activate GCN2 by binding to a histidyl-tRNA synthetase-related domain located C-terminal to the kinase domain . S. cerevisiae GCN2 is activated not only by nutrient limitation including amino acid, purine and glucose deprivation, but also by high concentration of sodium and by the immunosuppressant rapamycin .
In plants, the lower dissociation constant of eIF2 for GDP compared to yeast or mammals have led several years ago to the assumption that plants do not use phosphorylation of eIF2α as a mean to regulate translation . Furthermore, the observation that starvation for aromatic or branched amino acids did not initiate a general response have also led to the suggestion that, in contrast to yeast, such general amino acid response may not exist in plants . Therefore the physiological significance of eIF2α phosphorylation in plants is largely unknown. More recently, a GCN2-like enzyme (AtGCN2) has been identified in Arabidopsis thaliana and was shown to restore the growth of a yeast gcn2 mutant in the presence of an inhibitor of amino acids biosynthesis . AtGCN2 was also shown to be activated in plants by amino acid deprivation conditions .
In this work, we show that in response to several stresses, including amino acid and purine starvations, UV, cold shock and wounding, Arabidopsis GCN2 kinase is activated and phosphorylates eIF2α. We show that GCN2 is essential for plant growth in stress situations and that this activity is linked to a strong reduction in global protein synthesis. The activation of GCN2 following wounding or exposure to methyl jasmonate, ACC and salicylic acid, further suggests that this enzyme could play a role in plant defense responses to insect herbivores and may represent a key but yet uncharacterized player linking biotic and abiotic stresses.
Results and discussions
Arabidopsisand rice have a single eIF2α kinase
In addition to the presence of a GCN2 enzyme, higher plants have been proposed to contain a double-stranded RNA-dependent protein kinase with biochemical properties of the mammalian PKR [13–15]. Using the protein kinase domain of the yeast and Arabidopsis GCN2 and of the human PKR, PERK and HRI enzymes, we searched the Arabidopsis genome for the presence of additional eIF2α kinases. GCN2, PRK, PERK and HRI protein kinase domains were used in a BLASTP analysis against the TAIR 8.0 Arabidopsis proteins http://www.arabidopsis.org and, in each case, fifty proteins with the highest E value were collected. Using this approach, we produced a list of 138 non-redundant kinases (out of the more than 1000 Arabidopsis kinases) that most closely resemble eIF2α kinases (Additional file 1). Using a phylogenetic approach, we compared these 138 kinase domains to yeast, human and mouse eIF2α kinase domains. Two Arabidopsis kinases were found to form a statistically significant cluster with known eIF2α kinases (Additional file 1): one is AtGCN2 (AT3G59410) while the other is annotated as a Wee1-like kinase (AT1G02970) . To evaluate the capacity of the Wee1-like protein to bind to eIF2α, we analyzed in more detail the alignment of this enzyme to the other eIF2α kinases (Additional file 2). We observed that three essential structural characteristics of eIF2α kinases are not present in the Wee1-like kinase: a large insert (more than 30 amino acids) between the conserved kinase domains IV and V , a threonine in a putative autophosphorylation site (position 446 for the human PKR)  and a threonine at the end of domain IX (position 487 for the human PKR) that is critical for the substrate specificity . Therefore, although the Arabidopsis Wee1-like enzyme possess a high degree of homology to eIF2α kinases, as previously noted for the human Wee1 , it is unlikely to bind to eIF2α. We conclude that Arabidopsis likely possesses a single eIF2α kinase of the GCN2 type. The same conclusion was obtained using this approach to analyze the rice genome (data not shown) suggesting that higher plants only contain a GCN2-like eIF2α kinase.
Arabidopsisthaliana GCN2 can phosphorylate eIF2α and is essential in amino acid deprivation conditions
In a classical rich medium, gcn2 mutant plants do not have a particular phenotype compared to wild-type plants. However, we observed that gcn2 plants are much more sensitive to chlorsulfuron herbicide treatment than wild-type plants (data not shown). Following 6 days of chlorsulfuron-induced amino acid starvation gcn2 seedlings appeared smaller and more chlorotic than wild-type seedlings, a result that concurs with that of Zhang et al. . The introduction of an intact copy of AtGCN2 in the gcn2 mutant line allows the plants to grow identically to wild-type seedlings during amino acid deprivation (data not shown), confirming that AtGCN2 is essential for plant growth in this stress situation. As expected, rescue of eIF2α phosphorylation in response to amino acid deprivation was also observed in the complemented gcn2:GCN2 plants (Figure 2). These results confirm that AtGCN2 is the only Arabidopsis kinase involved in eIF2α phosphorylation in amino acid starvation conditions.
Characterization of other stresses leading to eIF2α phosphorylation in Arabidopsis
The AtGCN2 activity is not regulated by the TOR pathway
GCN2 activity induces a strong reduction in global protein synthesis
Our data indicate that AtGCN2 is important for plant growth in stress situations, likely through the general down regulation of mRNA translation. For the moment, it is not known if eIF2α phosphorylation by AtGCN2 can lead to the selective stimulation of specific mRNA translation like this is the case for the yeast GCN4  or mammalian ATF4 mRNAs . The strong activation of AtGCN2 following wounding and exposure to key hormones suggests that this enzyme evolved to play a role in plant defense responses to insect pathogens and may represent a key but yet uncharacterized player linking biotic and abiotic stresses. Therefore, while mammalians use four different eIF2α kinases to respond to a large variety of biotic and abiotic stresses, plants apparently achieve a similar result with a single, GCN2-like enzyme.
Plant material, growth condition and stress treatments
Wild-type and gcn2 mutated Arabidopsis thaliana plants are from the Landsberg (ler-0) ecotype. The gcn2 insertion line was obtained from RIKEN Genomic Science Center http://www.riken.jp/engn. Sterile seeds were germinated on solid Murashige and Skoog (MS) medium containing 1% sucrose (w/v) and cultured in a 16-h-light/8-h-dark cycle for 10 days at 23°C. When appropriate, Chlorsulfuron, glyphosate, 8-azaadenine, methyl jasmonate, salicylic acid, ACC and branched amino-acid were added to the medium at a final concentration of 0.6 μM, 1.5 mM, 50 μg/ml, 25 μM, 0.6 mM, 50 μM and 5 mM respectively. For the UV stress, seedlings were irradiated with UV light at 50 mJ/m2 by using a UV cross-linker (Bio-Rad Laboratories, Inc.). Plant wounding was performed by crushing the leaf across the midrib with a haemostat. For the cold shock treatment, seedlings were transferred at 4°C during 2 h followed by 2 h back in the growth chamber at 23°C. Heat shock were performed at 42°C during 2 h followed by 2 h back in the growth chamber at 23°C. For osmotic and oxidative stresses, the seedlings were placed in solutions containing 250 mM NaCl or 1 mM H2O2 during the time indicated.
Immunoblot analysis of eIF2α phosphorylation
Western blots were probed using a phosphospecific anti-eIF2α rabbit monoclonal antibody (Epitomics, Burlingame, CA; 1/1,000 dilution). After incubation with a horseradish peroxydase-coupled anti-rabbit secondary antibody (Sigma 1/5,000 dilution) immunoblots were developed by using the ECL Plus Western Blotting detection reagents (GE Healthcare Bio-Sciences). Chemiluminescence was visualized with a VersaDoc Imaging System (Bio-Rad Laboratories, Inc.). Equal loading of protein were confirmed by reprobing the membranes with a mouse monoclonal anti-alpha-tubulin (Sigma 1/5,000 dilution).
Polysome preparation and detection of neo-synthesized proteins in normal and stress situation
For polysome preparation, seeds were sown in liquid medium, incubated 48 h at 4°C, and grown under constant light during 10 days at 23°C. Chlorsulfuron was added, and after 2 or 4 hours, the ribosomal pellet fraction was prepared from wild-type (WT) or gcn2 mutant seedlings as described in . Polysome profiles were displayed on sucrose gradients and profiles recorded at 260 nm. For the detection of neo-synthesized proteins, the 10 days-old seedlings were treated for 2 to 4 hours with 8-azaadenine and then transferred for 2 hours into 1 ml of MS medium containing 50 μCi of 35S-labelled methionine and 8-azaadenine. Untreated controls were incubated for 2 hours into the same labelling medium, but in the absence of 8-azaadenine. After two washes with MS medium, total proteins were extracted as described below and 35 μg of protein were separated on SDS-PAGE. Gels were stained with Coomassie blue or dried and autoradiographied using a PhosphoImager (Bio-Rad Laboratories, Inc.).
Preparation of total protein extracts
Following exposure to stress, total protein extracts were prepared as following. For each treatment, three seedlings were ground in Laemmli buffer containing both Complete protease and PhosSTOP phosphatase inhibitor (Roche Diagnostics, Indianapolis, IN) and incubated at 95°C for 5 min. After centrifugation, samples were fractioned by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose.
This work was supported by the CNRS and the Université of Perpignan.
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