eIFiso4G Augments the Synthesis of Specific Plant Proteins Involved in Normal Chloroplast Function

Abstract

The plant-specific translation initiation complex eIFiso4F is encoded by three genes in Arabidopsis (Arabidopsis thaliana)-genes encoding the cap binding protein eIFiso4E (eifiso4e) and two isoforms of the large subunit scaffolding protein eIFiso4G (i4g1 and i4g2). To quantitate phenotypic changes, a phenomics platform was used to grow wild-type and mutant plants (i4g1, i4g2, i4e, i4g1 x i4g2, and i4g1 x i4g2 x i4e [i4f]) under various light conditions. Mutants lacking both eIFiso4G isoforms showed the most obvious phenotypic differences from the wild type. Two-dimensional differential gel electrophoresis and mass spectrometry were used to identify changes in protein levels in plants lacking eIFiso4G. Four of the proteins identified as measurably decreased and validated by immunoblot analysis were two light harvesting complex binding proteins 1 and 3, Rubisco activase, and carbonic anhydrase. The observed decreased levels for these proteins were not the direct result of decreased transcription or protein instability. Chlorophyll fluorescence induction experiments indicated altered quinone reduction kinetics for the double and triple mutant plants with significant differences observed for absorbance, trapping, and electron transport. Transmission electron microscopy analysis of the chloroplasts in mutant plants showed impaired grana stacking and increased accumulation of starch granules consistent with some chloroplast proteins being decreased. Rescue of the i4g1 x i4g2 plant growth phenotype and increased expression of the validated proteins to wild-type levels was obtained by overexpression of eIFiso4G1. These data suggest a direct and specialized role for eIFiso4G in the synthesis of a subset of plant proteins.

Figure 1.

Plant high throughput phenotyping of single, double, and triple mutants. A, Projected leaf area of Arabidopsis lines grown under low, normal, or high white light conditions. Note that the scale differs for each light level. Statistical analysis P-value adjustment used the Tukey test for n = 15 and had a 0.05 significance level. Error bars = (se). B, Representative images of the size difference of Arabidopsis lines grown in soil (normal light, 16 h light) and photographed at 30-d postgermination.

Figure 2.

2D-DIGE of proteins extracted from double or triple mutants. Protein extracts were cy2 (Col-0) or cy3 (i4g1 x i4g2 or i4f) dye-labeled and run on 2D-PAGE (Applied Biomics). A, Wild type (Col-0) and mutant (i4g1 x i4g2; i4f) 2D gels are shown. B, Superimposed images comparing wild-type and mutant protein extracts as indicated. Green indicates the protein is decreased relative to Col-0, red indicates the protein is increased relative to Col-0, and yellow indicates that the protein level remained the same. Proteins that were measurably increased or decreased were identified by mass spectrometry. See Supplemental Table S1 for all mutants.

Figure 3.

Confirmation by western blotting of protein targets identified as decreased by 2D-DIGE in double or triple mutants. Total plant extracts were probed with antibodies to protein targets identified by 2D-DIGE in wild-type and mutant plants. A, Proteins that were the most decreased evidenced by 2D-DIGE: Lhcb3, Lhcb1, RCA, and CA1; i4G and i4E and actin are included as controls. B, Additional proteins identified as decreased in the 2D-DIGE: PsbP, VIPP1, PsbQ, and PsbO. See Supplemental Figure S4A for an example of the Stain-Free gel for protein loading comparison. MW, molecular weight.

Figure 4.

Photosynthetic efficiency of single, double, and triple mutants. A, Measurements of PSII photosynthetic efficiency in 21-d–old wild-type and mutant plant lines as determined by chlorophyll a fluorescence induction: QY_max, ABS/RC, TRo/RC, ETo/RC, ETo/TRo, and DIo/RC. Error bars = true mean ± se (n = 15–16) and asterisks indicate measurements that passed a two-tailed t test with P value < 0.01. B, OJIP fluorescence transient observed for the same lines (average curves normalized to F_O and F_M). C, Western blot of CYP38. See Supplemental Figure S4B for Stain-Free gel for loading control. MW, molecular weight.

Figure 5.

TEM of wild-type and i4g1 x i4g2 double mutant chloroplasts. A to F, Cross sections of Col-0 (A–C) and i4g1 x i4g2 double mutant plants (D–F). Scale bars = 1 μm (A and D), 0.5 μm (B and E), or 0.2 μm (C and F). Mutant chloroplasts show more diffuse grana with fewer stacks and a higher incidence of starch granules (see Supplemental Table S2).

Figure 6.

Protein stability analysis of proteins in the i4g1 x i4g2 double mutant. Protein extracts were prepared from tissue treated with CHX to inhibit new translation. Extracts of Col-0 and the i4g1 x i4g2 double mutant were compared with and without CHX treatment. Antibodies were used to detect Lhcb3, Lhcb1, RCA, CA1, i4G and i4E, and actin. HA-tagged IAA1 is used as a control for stability. See Supplemental Figure S4C for Stain-Free gel for loading control. MW, molecular weight.

Figure 7.

Effect of CaMV 35S overexpression of eIFiso4G1 in i4g1 xi 4g2 plants. A, 30-d–old plants grown in soil: Col-0 wild type, i4g1 xi 4g2 mutant, A1, E1, and F2 independent lines overexpressing eIFiso4G1 (16-h light). B, Western blot showing effect of overexpression of eIFiso4G1 with antibodies to Lhcb3, Lhcb1, RCA, CA1, i4E, i4G1, cyclophilin 38 (CYP38), and actin. See Supplemental Figure S4D for Stain-Free gel for loading control. MW, molecular weight.