Deletion of the eIFiso4G subunit of the Arabidopsis eIFiso4F translation initiation complex impairs health and viability

Abstract

Arabidopsis thaliana knockout lines for the plant-specific eukaryotic translation initiation factors eIFiso4G1 (i4g1) and eIFiso4G2 (i4g2) genes have been obtained. To address the potential for functional redundancy of these genes, homozygous double mutant lines were generated by crossing individual knockout lines. Both single and double mutant plants were analyzed for changes in gross morphology, development, and responses to selected environmental stressors. Single gene knockouts appear to have minimal effect on morphology, germination rate, growth rate, flowering time, or fertility. However, double mutant i4g1/i4g2 knockout plants show reduced germination rates, slow growth rates, moderate chlorosis, impaired fertility and reduced long term seed viability. Double mutant plants also exhibit altered responses to dehydration, salinity, and heat stress. The i4g2 and i4g1/i4g2 double mutant has reduced amounts of chlorophyll a and b suggesting a role in the expression of chloroplast proteins. General protein synthesis did not appear to be affected as the levels of gross protein expression did not appear to change in the mutants. The lack of a phenotype for either of the single mutants suggests there is considerable functional overlap. However, the strong phenotypes observed for the double mutant indicates that the individual gene products may have specialized roles in the expression of proteins involved in plant growth and development.

Fig. 1

a eIF4G and eIFiso4G Domain Organization. The organization of eIF4G from vertebrates, fungi/yeast and plants have in common the eIF4E binding domain and core HEAT domain for binding eIF4A and eIF3 (MIF4G). Fungi and yeast have lost the second and third HEAT domains that include the second eIF4A binding domain and MNK binding domain (Hernandez and Vazquez-Pianzola ; Marintchev and Wagner ; Schutz et al. 2008). Plant eIF4G and eIFiso4G have retained only the first and second HEAT domains. The PABP binding domain for plant eIF4G or eIFiso4G (***) has not been experimentally determined. b Amino Acid Sequence Comparison. The amino acid sequences for Arabidopsis thaliana eIF4G (At3g60240) and the two eIFiso4G (At5g57870, At2g24050) proteins were aligned using CLUSTALW2 (Larkin et al. 2007) and visualized with MACAW (v.2.0.5) (Schuler et al. 1991). The approximate binding domains for eIF4E/iso4E, eIF4A and eIF3 are indicated. The actual sequence alignments are shown in Supplemental Data Figure B. The PABP binding domain (***) for plant eIF4G or eIFiso4G has not been experimentally determined

Fig. 2

eIF4G/eIFiso4G Phylogeny. Amino acid sequences from the conserved HEAT-1/MIF4G domain were aligned for 50 eIF4G or eIFiso4G sequences from plants (vascular and non-vascular), animals, and fungi using the program MAFFT (Katoh and Toh 2008). A phylogenetic analysis of these aligned sequences (see Supplementary data Fig. C) was then conducted using PAUP* (Swofford 2000), under the parsimony, minimum evolution, and maximum likelihood criteria (the parsimony results are shown here), with bootstrapping used to assess support. Asterisks indicate >70% (*), >95% (**), and >99% (***) bootstrap support

Fig. 3

Arabidopsis eIFiso4G knockout lines. a Genomic screening. i4g1 lines carry a pROK2 T-DNA insertion in exon 9. i4g2 lines contain a pROK2 T-DNA insertion in exon 4. PCR screening of genomic DNA with gene specific primers yields a 928 bp band which is absent in homozygous i4g1 plants, but present in both wild-type Col-0 and heterozygous plants. Screening with a combination of pROK2 specific and gene specific primers yields a band of 601 bp in i4g1 and heterozygous plants that is absent in Col-0. PCR screening of genomic DNA with gene specific primers yields a 915 bp band which is absent in homozygous i4g2 plants, but present in both wild-type Col-0 and heterozygous plants. Screening with a combination of pROK2 specific and gene specific primers yields a band of 753 bp in i4g2 and heterozygous plants that is absent in Col-0. b Confirmation of eIFiso4G isoform knockout by RT–PCR and western blotting. mRNA populations from wild-type and mutant plants were probed for actin, eIFiso4G1, and eIFiso4G2 message (left panel). Western blotting of total plant extracts probed with antibody to AteIFiso4G1 (right panel upper). Coomassie stained Rubisco is shown for a total protein loading comparison (right panel lower). c Phenotypic effects. (Left panel) Overhead comparison of plants at 21 days post sowing. (Right panel) Profile of plant lines at ~56 days post sowing

Fig. 4

Confocal imagery of epidermal cells. Propidium iodide stained epidermal cells of 21 day old leaves at 20× magnification. A 450 μm × 450 μm section is shown

Fig. 5

Salinty responses. Root length and fresh weight was measured following transfer of 4 day old seedlings to new media containing 0 mM or 100 mM NaCl. Aseptically grown seedlings that were transplanted onto 100 mM NaCl media were weighed and compared to seedlings transferred to 0 mM NaCl media

Fig. 6

Thermotolerance. Hypocotyl lengths were measured following treatment at elevated temperatures. Plants were treated at room temperature, 37°C for 90 min, 45°C for 90 min, or 45°C for 60 min following pretreatment at 37°C for 90 min and 22°C for 60 min

Fig. 7

Incorporation of [³⁵S]-methionine in vivo. Leaves of seedlings (12 days) were labeled with [³⁵S]-methionine for 6 or 12 h (room temperature, continuous light) as described in Experimental Procedures