Dissecting the role of eIF2alpha phosphorylation in translational control using a transgenic plant model.

摘要

Eukaryotic initiation factor 2 alpha subunit (eIF2alpha) is involved in one of the rate-limiting steps for protein synthesis. The role of eIF2alpha in protein synthesis initiation has been demonstrated in many eukaryotes but not in plants. For this study, modification of the eIF2alpha phosphorylation pathway was attempted by expression of wheat eukaryotic initiation factor 2 alpha subunit wild type (weIFalpha51S), a dominant negative mutant of weIFalpha51S (weIFalpha51A), a double stranded RNA binding protein from vaccinia virus (E3L), human ds-RNA dependent protein kinase (hPKR), and a dominant negative mutant hPKR (hPKRK296R) in planta . In Nicotiana benthamiana, transient expression of weIFalpha51A, E3L, hPKR, or hPKRK296R, but not weIFalpha51S caused cell death based on visual analysis. Western blots revealed recombinant protein expression of the target genes except weIF2alpha51A for both estradiol induced and non-induced treatments indicating leaky gene transcription. Floral dipping transformation of Arabidopsis resulted in transformation efficiencies of 0.95 to 1.57 percent in the T1 generation. Phenotype analysis of the T3 generation revealed that plants putatively transformed with weIF2alpha51S were taller than those with weIF2alpha5IA or the empty plasmid control (pXVE:2HA). The plants putatively transformed with E3L were shorter and had reduced leaf numbers per plant compared to the control (pXVE). The plants putatively transformed with hPKR and hPKRK296R were shorter than those with pXVE. Plants putatively transformed with hPKR K296R had greater leaf numbers per plant compared with hPKR putatively transformed plants and the control. All plant lines studied were confirmed for T DNA insertion by PCR except for two plant lines putatively transformed with hPKRK296R. Gene-specific mRNA was detected from only two plant lines of weIF2alpha51S. However, there was no protein expression detected for any of the study genes. Results indicate that the plasmids pXVE:2HA and pXVE did not work as expected. Additional work is needed to define the role of eIF2alpha phosphorylation on plant growth and development.