Abwao, Stephen Indieka
Translational control of abiotic stress responses in Arabidopsis thaliana.
PhD thesis, University of Glasgow.
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A detailed understanding of the mechanisms by which plants sense and respond to major environmental stress factors will significantly contribute towards the prospects of developing crops capable of yielding well over a wider geographical range, including marginalised lands. One of the important stress response mechanisms in eukaryotes is mediated through phosphorylation of the eIF2α-subunit (serine 51/56) by specific kinases, namely double stranded RNA activated protein kinase (PKR), General Control Non-repressible 2 protein kinase (GCN2), Pancreatic eIF2α kinase (PERK) and Heme-regulated inhibitor protein kinase (HRI). This mechanism is a highly conserved phenomenon in eukaryotes and occurs in response to various stress conditions. Unlike in yeast and mammals, the mechanism is however not well established in higher plants, although its components such as eIF2α and GCN2 kinase have been identified in plants. The objective of the study reported herein was therefore to elucidate this mechanism in Arabidopsis, a model plant species.
Initially the presence of yeast GCN2 kinase (ScGCN2), human PKR (HsPKR), human HRI (HsHRI) and human PERK (HsPERK) kinase homologues in Arabidopsis and Viridiplantae (green plants and algae) was evaluated through homology and phylogenetic analysis using TAIR10 and NCBI protein sequences, respectively. Arabidopsis lacked homologues of HsPKR, HsHRI and HsPERK however the presence of ScGCN2 homologue, herein referred to as AtGCN2 (Arabidopsis GCN2 kinase), was confirmed. Further evaluation of translation control mechanism through phosphorylation of AteIF2α (Arabidopsis eukaryotic initiation factor 2 α-sub-unit) was conducted using Atgcn2-1 null mutant plants (plants expressing a copy of truncated non-functional AtGCN2 kinase). Unlike WT Col-0, the Atgcn2-1 seedlings failed to induce phosphorylation of AteIF2α after exposure to amino acid starvation (150 µM glyphosate), NaCl (50 and 100 mM), heat (37oC) and cold (4oC) acclimation. On the other hand no strong phenotype of Atgcn2-1 was observed under optimal growth conditions and NaCl stress, except seedlings had relatively shorter roots compared with WT Col-0 seedlings. Failure of Atgcn2-1 seedlings to induce phosphorylation of AteIF2α Ser 56, after exposure to various stress confirmed that Arabidopsis possesses only one GCN2 kinase, as is in the case of yeast, and unlike mammalian systems. Further characterisation was conducted by exposing WT Col-0, Atgcn2-1, jar-1 and NahG seedling to biotic stress; Cauliflower Mosaic Virus (CaMV) and Pseudomonas syringae DC3000 (P. syringae) and positive control treatment using 150 µM glyphosate. The jar-1 and NahG seedlings are mutants defective in jasmonate and salicylic pathways, respectively. Inoculation with CaMV and P. syringae failed to induce phosphorylation of AteIF2α, unlike glyphosate. These results suggested that activation of the AtGCN2 kinase may be independent of jasmonate and salicylic pathways.
Due to lack of a strong Atgcn2-1 phenotype, two mutants expressing AtGCN2 under the control of a 35S promoter, namely p35S:AtGCN2 and p35S:GFP:AtGCN2 were generated for further characterisation and localisation of AtGCN2 kinase, respectively. For characterization experiments the p35S:AtGCN2 seedlings were subjected to salinity stress, osmotic stress and temperature shock. In localisation experiments, GFP activities were assessed in non-stressed 7-day old p35S:GFP:AtGCN2 seedlings. During characterisation, higher germination rates were generally obtained with p35S:AtGCN2 and Atgcn2-1 compared with WT Col-0 seeds on ½ MS media containing NaCl, KCl and mannitol. On media infused with PEG6000 however p35S:AtGCN2 had the lowest germination rates. There were also no strong p35S:AtGCN2 phenotypes observed, except for increased root growth compared with WT Col-0 and Atgcn2-1 seedlings. In contrast, Atgcn2-1 seedlings subjected to PEG6000 osmotic stress had the highest increase in root growth compared with both WT Col-0 and p35S:AtGCN2 seedlings. On the other hand localisation of the GFP:AtGCN2 fusion protein was observed in the root and shoot tip tissues of p35S:GFP:AtGCN2 seedlings. The results obtained with Atgcn2-1 and p35S:AtGCN2 seedlings suggested that mutation of Atgcn2 produced root phenotypes. There were no significant differences in the survival of all the three genotypes when seedlings were subjected to heat shock stress. In cold shock experiments however Atgcn2-1 survival was significantly (p<0.05) lower than that of WT Col-0 and p35S:AtGCN2 seedlings, thus suggesting that null mutation of Atgcn2 increased susceptibility of seedlings to cold shock.
The homologues of the yeast General Control Non-repressible 4 (ScGCN4) and human Activating Transcriptional Factor 4 (HsATF4), that are activated, when yeast and mammalian eIF2α is phosphorylated, respectively are yet to be identified in plants. To identify putative Arabidopsis ScGCN4 and HsATF4 homologues both in vitro and in silico approaches were explored. In vitro translation experiments using Wheat Germ Lysate (WG) mimicking plant translation under stress (WGeIF2α-P) and non-stress (WGeIF2α) conditions were conducted. To mimic stress conditions mPKR kinase was added into the translation reaction and significantly inhibited protein synthesis compared to control treatment. However, due to technical difficulties it was not possible to identify all translated transcripts under stress conditions (WGeIF2α-P) thereby identifying potential Arabidopsis ScGCN4 and HsATF4 homologues. This prompted the use of in silico tools to identify putative Arabidopsis homologues of ScGCN4 and HsATF4 using the FivePrime Viewer programme (Webb, 2008). A total of 99 TAIR10 transcripts with 5′ upstream Open Reading Frames (uORFs) were identified and only two transcripts, AT4G31590.1 and AT1G58120.1, were identified as putative homologues of ScGCN4 and non for HsATF4. The AT4G31590.1 and AT1G58120.1 transcripts encode for proteins involved in cellulose synthase/ gylcosyl transferase and methyl transferase activities, respectively. Although these genes are involved in key plant growth and developmental activities, there is need to assess translation control of their main open reading frame (mORF) by uORFs through phosphorylation of AteIF2α.
Overall the data presented in this study suggest that stress response translation regulation mechanism mediated by phosphorylation of eIF2α is present in Arabidopsis. Plants are known, however, to carry out unique biological processes such as photosynthesis and cellulose biosynthesis that other eukaryotes lack. It would therefore not be surprising for them to have translation regulation mechanisms like other eukaryotes but with unique differences.
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