Figure 1.
Defects of eif3h mutant Arabidopsis in shoot apical meristem maintenance.
(A–B) 12 day old SAM imaged by differential interference contrast after clearing with chloral hydrate; arrows point to SAM. (A) wild type. (B) eif3h mutant. (C–D, H) Scanning electron micrographs of 3 week old wild-type inflorescence meristem (C) and equivalent in eif3h (D and H). Note enlargement of cells and of the entire SAM in eif3h. Arrow points to the SAM. The meristem in (D) is fasciated, i.e. branched into two, and both (D) and (H) have formed a radialized leaf. (E–G) Enlarged shoot apex in eif3h. White arrows point to the inflorescence (in wild type) or the shoot apex (in eif3h). (E) Wild type. (F) The enlarged quiescent eif3h SAM (inset shows a close-up of the apex). (G) Further enlarged dome-shaped eif3h SAM prior to senescence (inset shows a close-up). (I) Reactivated eif3h SAM with multiple apices initiating. (J–M) Filamentous organs emerging from the eif3h apex (arrows). (J) 3 week old eif3h mutant. (K) Filamentous organ with trichomes on 3 week old eif3h apex. (L) Filamentous organ without trichomes on 3 week old eif3h apex. (M) A filamentous leaf on a 1 week old eif3h apex. (N–P) Inflorescence apices. Arrows point to the SAM. (N) Wild type. (O) eif3h. (P) 3-week old clv3-2.
Table 1.
Meristem abnormalities in the eif3h mutant.
Figure 2.
The 5′ leader of the CLV1 mRNA renders translation dependent on eIF3h.
(A) The 5′ leader of CLV1 harbors multiple uORFs. The boxes stand for uORFs that are in the –1 frame (green), or in the +1 frame (blue) with the main ORF. The cDNA sequence corresponds to the longest known gene model: CLV1 (At1g75820.1). (B) Schematic view of the mRNAs for protoplast transformation. mRNAs were prepared by in vitro transcription with SP6 RNA polymerase. An equal amount of internal control (Spacer-LUC+) mRNA was added to the 5′ leader-RLUC mRNA to be tested as an internal control for transformation efficiency. (C) Translational efficiency on the CLV1 and WUS 5′ leader is expressed as the mean RLUC/FLUC ratio with standard errors from three replicate transformations.
Table 2.
Polysome loading state of selected mRNAs in eif3h mutant versus wild type.
Figure 3.
Translation assays with reference gene driven by the crTMV intergenic sequence.
(A) Structure of the expression plasmids. In A1, the experimental FLUC reporter gene is transcribed by the 35S promoter and harbors the 5′ leader to be tested for translational efficiency. The RLUC reference gene is located further downstream on the same plasmid and is expressed due to transcriptional promoter activity of the crTMV sequence element. In A2, the experimental reporter is RLUC and the reference is LUC+. (B–I) Plasmids were transiently transformed into 10 day old wild-type or eif3h-1 seedlings. The expression is given as the ratio of the reporter luciferase activity divided by the reference luciferase from three replicate transformations with standard error. (B) The A1 construct with or without the crTMV sequence was used to confirm that the crTMV element is not eIF3h dependent. In this exceptional case, data are shown as downstream : upstream activity. (C–H) Tests of four plant 5′ leaders. (C) AtbZip11; (D) HY5; (E) HY5 leader in the RLUC-LUC+ construct (A2). AtbZip11 and HY5 leaders served to evaluate the translation assay system. (F, G) PIN1; (H) CLV1; (I) CLV1 uORF-less. HY5 has only one very short uORF and PIN1 has none.
Figure 4.
Removal of uORFs from the CLV1 5′ leader reduces its eIF3h dependence.
(A) Transient dual luciferase assays were performed in protoplasts as in Figure 2 but data are presented as the relative expression in the eif3h mutant as compared to wild type with standard deviations. Asterisks represent up to five uAUGs and open boxes represent uORFs. (B) Stable transgenic plants harboring the dual-luciferase construct illustrated at the top. The CLV1 native leader or uORF-less leader is linked to FLUC, and the RLUC ORF driven by the crTMV-element serves as a reference. Translational efficiency of the 5′ leader was established via dual luciferase assays in 10-day-old seedlings from three independent transgenic lines. Statistical significance was determined by t-test (***for p-value <0.001 and **for P-value<0.05). x0.05: FLUC activities of one particularly highly expressing line were multiplied by 0.05 for convenience of display.
Figure 5.
WUS and CLV3 expression in the eif3h mutant.
(A) Reverse transcription (RT) PCR results for WUS and CLV3 mRNAs from 2 week old plants along with translation elongation factor 1α as a control. In eif3h, two CLV3 transcripts corresponding to gene models At2g27250.1 and At2g27250.3 were detected. To detect the transcripts in wild type, more amplification cycles are needed. (B–D) WUS:GUS expression [57] in wild type (B) and eif3h (C, D) seedlings. (E–G) CLV3:GUS expression in wild type (E) and eif3h (F, G) seedlings. (E) is at half the magnification of F and G. (H, I) WUS:GUS expression in wild type (H) and eif3h (I) inflorescences. (J, K) WUS:GUS expression in wild type (J) and eif3h (K) flowers. (L, M) CLV3:GUS expression in the wild type (L) and eif3h (M) inflorescences. (N, O) CLV3:GUS expression in wild type (N) and eif3h (O) flowers. (P) shows elevated CLV3:GUS expression in stamens and petals of a developing eif3h flower bud.
Figure 6.
Reduced translation behind the ASYMMETRIC LEAVES 1 leader in the eif3h mutant, and defects on leaf polarity in the eif3h mutant and eif3h/as2 or eif3h/as1 double mutants.
(A) Translation assay results for the AS1 (At2g37630.1) leader and its uORF-removed variants. uAUGs in the AS1 leader were mutated as described in material and methods for removing uAUGs from the CLV1 leader. The translation assay was performed as in Figure 2 and data analyzed as in Figure 4. Statistical significance was determined by t-test (**for p-value <0.05). (B) Rosette leaves of eif3h, as1 and as2 (At1g65620) mutants. (C–E) Scanning electron microscopic images for eif3h rosette leaves with outgrowth on the abaxial side. Arrows point to outgrowths without (C and D) or with (E) trichomes. (F–G) Rosette leaves and needle-like leaf of the eif3h/as2 double mutant with additional leaflets. Arrows point to an expanded leaflet (F), needle-like leaflets (G) and needle-like leaflets on a needle-like leaf. (I–L) Ectopic structures growing on the eif3h/as1 double mutant rosette leaves. Arrows point to an ectopic shoot growing (I) and SEM images of ectopic ovule like structures (J, K, L).
Figure 7.
A concept map for the role of eIF3h in Arabidopsis SAM maintenance and auxin response.
By overcoming the translational repression by uORFs, eIF3h promotes the translation of ARFs [16] and CLV1 and AS1 (this work), and therefore plays an important role in SAM maintenance and organogenesis.