The 5′ leader of the CLV1 mRNA renders translation dependent on eIF3h.

<p>(<b>A</b>) The 5′ leader of <i>CLV1</i> 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: <i>CLV1</i> (At1g75820.1). (<b>B</b>) Schematic view of the mRNAs for protoplast transformation. mRNAs were prepared by <i>in vitro</i> 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. (<b>C</b>) Translational efficiency on the <i>CLV1</i> and <i>WUS</i> 5′ leader is expressed as the mean RLUC/FLUC ratio with standard errors from three replicate transformations.</p

Translation assays with reference gene driven by the crTMV intergenic sequence.

<p>(<b>A</b>) 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>B–I</b>) Plasmids were transiently transformed into 10 day old wild-type or <i>eif3h-1</i> 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>B</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. (<b>C–H</b>) Tests of four plant 5′ leaders. (<b>C</b>) <i>AtbZip11;</i> (<b>D</b>) <i>HY5</i>; (<b>E</b>) <i>HY5</i> leader in the RLUC-LUC+ construct (A2). <i>AtbZip11</i> and <i>HY5</i> leaders served to evaluate the translation assay system. (<b>F, G</b>) <i>PIN1</i>; (<b>H</b>) <i>CLV1;</i> (<b>I</b>) <i>CLV1</i> uORF-less. <i>HY5</i> has only one very short uORF and <i>PIN1</i> has none.</p

Removal of uORFs from the <i>CLV1 5′</i> leader reduces its eIF3h dependence.

<p>(<b>A</b>) Transient dual luciferase assays were performed in protoplasts as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095396#pone-0095396-g002" target="_blank"><b>Figure 2</b></a> but data are presented as the relative expression in the <i>eif3h</i> mutant as compared to wild type with standard deviations. Asterisks represent up to five uAUGs and open boxes represent uORFs. (<b>B</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.</p

A concept map for the role of eIF3h in <i>Arabidopsis</i> SAM maintenance and auxin response.

<p>By overcoming the translational repression by uORFs, eIF3h promotes the translation of <i>ARF</i>s <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095396#pone.0095396-Zhou1" target="_blank">[16]</a> and <i>CLV1</i> and <i>AS1</i> (this work), and therefore plays an important role in SAM maintenance and organogenesis.</p

Reduced translation behind the <i>ASYMMETRIC LEAVES 1</i> leader in the <i>eif3h</i> mutant, and defects on leaf polarity in the <i>eif3h</i> mutant and <i>eif3h/as2</i> or <i>eif3h/as1</i> double mutants.

<p>(<b>A</b>) Translation assay results for the <i>AS1</i> (At2g37630.1) leader and its uORF-removed variants. uAUGs in the <i>AS1</i> leader were mutated as described in material and methods for removing uAUGs from the <i>CLV1</i> leader. The translation assay was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095396#pone-0095396-g002" target="_blank"><b>Figure 2</b></a> and data analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095396#pone-0095396-g004" target="_blank"><b>Figure 4</b></a>. Statistical significance was determined by t-test (**for p-value <0.05). (<b>B</b>) Rosette leaves of <i>eif3h</i>, <i>as1</i> and <i>as2</i> (At1g65620) mutants. (<b>C–E</b>) Scanning electron microscopic images for <i>eif3h</i> rosette leaves with outgrowth on the abaxial side. Arrows point to outgrowths without (<b>C</b> and <b>D</b>) or with (<b>E</b>) trichomes. (<b>F–G</b>) Rosette leaves and needle-like leaf of the <i>eif3h/as2</i> double mutant with additional leaflets. Arrows point to an expanded leaflet (<b>F</b>), needle-like leaflets (<b>G</b>) and needle-like leaflets on a needle-like leaf. (<b>I–L</b>) Ectopic structures growing on the <i>eif3h/as1</i> double mutant rosette leaves. Arrows point to an ectopic shoot growing (<b>I</b>) and SEM images of ectopic ovule like structures (<b>J, K, L</b>).</p

Polysome loading state of selected mRNAs in <i>eif3h</i> mutant versus wild type.

<p>Note: TL <i>eif3h</i>/WT. Translation state (TL) is the ratio of mRNA in polysomal and non-polysomal RNA fractions; it is log-transformed and unitless. When compared between <i>eif3h</i> mutant and wild-type seedlings, negative numbers indicate that the mRNA is less polysomal, i.e. undertranslated in the <i>eif3h</i> mutant. The values are means from duplicate polysome microarrays <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095396#pone.0095396-Kim1" target="_blank">[13]</a>. TOADSTOOL and the heterodimer of CLV2 and CORYNE are receptor like kinases that can function as CLV3 receptors in parallel to CLV1. Data for <i>ARFs</i>, <i>TIR1</i> and <i>AUX1</i> are included for the purpose of calibration, given that <i>ARFs</i> are also undertranslated in <i>eif3h</i> while <i>TIR1</i> and <i>AUX1</i> are not <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095396#pone.0095396-Zhou1" target="_blank">[16]</a>. The median value from 8832 genes was −0.10±0.21. <i>TOADSTOOL</i> and <i>AS1</i> and <i>CLV1</i> rank 19th and 55th and 495th, respectively, among cytosolic mRNAs. uORF numbers are from TAIR10. NA, not available.</p

<i>WUS</i> and <i>CLV3</i> expression in the <i>eif3h</i> mutant.

<p>(<b>A</b>) Reverse transcription (RT) PCR results for <i>WUS</i> and <i>CLV3</i> mRNAs from 2 week old plants along with translation elongation factor 1α as a control. In <i>eif3h</i>, two <i>CLV3</i> 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>B–D</b>) <i>WUS:GUS</i> expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095396#pone.0095396-Brand2" target="_blank">[57]</a> in wild type (<b>B</b>) and <i>eif3h</i> (<b>C, D</b>) seedlings. (<b>E–G</b>) <i>CLV3:GUS</i> expression in wild type (<b>E</b>) and <i>eif3h</i> (<b>F, G</b>) seedlings. (<b>E</b>) is at half the magnification of F and G. (<b>H, I</b>) <i>WUS:GUS</i> expression in wild type (<b>H</b>) and <i>eif3h</i> (<b>I</b>) inflorescences. (<b>J, K</b>) <i>WUS:GUS</i> expression in wild type (<b>J</b>) and <i>eif3h</i> (<b>K</b>) flowers. (<b>L, M</b>) <i>CLV3:GUS</i> expression in the wild type (<b>L</b>) and <i>eif3h</i> (<b>M</b>) inflorescences. (<b>N, O</b>) <i>CLV3:GUS</i> expression in wild type (<b>N</b>) and <i>eif3h</i> (<b>O</b>) flowers. (<b>P</b>) shows elevated <i>CLV3:GUS</i> expression in stamens and petals of a developing <i>eif3h</i> flower bud.</p

Meristem abnormalities in the <i>eif3h</i> mutant.

1)<p>Determined in situ by differential interference microscopy from 22 (<i>eif3h</i>) and 52 (wild type) 12 day old seedlings.</p>2)<p>Determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095396#pone-0095396-g001" target="_blank"><b>Figure 1J</b></a> under a stereomicroscope.</p