DEV017913 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Analyses of the ae6 locus. (A) Diagrams of the 3′ region of the RPL5A gene (At3g25520). Black and red boxes show coding region and 3′ untranslated region of the gene, respectively. A horizontal line under the gene indicates the region from which the probe was synthesized for Southern hybridization. Arrows indicate primers used in PCR analyses (see below). Tag1 (Tagging for Arabidopsis genes1) is an Arabidopsis transposon, the presence of which in the RPL5A 3′ UTR in the ae6-1 mutant was verified by sequencing. (B) Southern blot showing differences in restriction sizes of genomic DNA between wild-type Ler and ae6-1. (C) PCR showing that the 3′ UTR of RPL5A was disrupted. (D) RT-PCR analyses showing that the steady-state transcript level of RPL5A declined markedly in the ae6-1 mutant. The primers used for generation of the probe for Southern analysis and PCR in A are as follows: p1, 5′-GTGATGATGATGATGAGGAC-3′; p2, 5′-CCCGGGATTGCAGCTGCAAGAGTACC-3′; p3, 5′-GATGAAGTCTAACAGTTCTTAC-3′; and p4, 5′-GCTCCAAGAGAATACTGAG-3′. The primers p1 and p3 were used for RT-PCR in D.

• Supplemental Figure S2 -

  Fig. S2. Transgenic complementation of the ae5-1 as2-101 and ae6-1 as2-101 mutants. Diagrams of the RPL28A (A) and RPL5A (B) complementation constructs. (C) An ae5-1 as2-101 seedling. (D) A T1 ae5-1 as2-101 transgenic plant carrying the RPL28A complementation construct. (E) An ae6-1 as2-101 seedling. (F) A T1 ae6-1 as2-101 transgenic plant carrying the RPL5A complementation construct. Scale bars: 5 mm in C-F.

• Supplemental Figure S3 -

  Fig. S3. Identification of insertional mutants. Gene structures of RPL28A, RPL5A and RPL5B. Arrows indicate the positions of primers used in genotyping. Gene-specific primers plus a T-DNA left-border primer LBA1 (5′-TGGTTCACGTAGTGGGCCATCG-3′) were used in genotyping, with genomic DNA as templates. Primers used are as follows: 28A-F, 5′-ATGGCGACAGTTCCAGGAC-3′ and 28A-R, 5′-TTAAGCTTGTCTGTTTCTTTG-3′ for ae5-2; 5A-F, 5′-CAACCAAGACAAGAACAAGTAC-3′ and 5A-R, 5′-TGTGAACACAAAAGCTGAGTG-3′ for ae6-2; 5B-F, 5′-CCATCTTTTGTCACTCATC-3′ and 5B-R, 5′-CCCATTTTCCACAAGCAC-3′ for rpl5b. Primers used for RT-PCR are as follows: 28A-F plus 28A-R for RPL28A; 5A-2F (5′-CACAATCTCACAGCAAAG-3′) plus 5A-R for RPL5A; 5B-2F (5′-GATGTACAAGAAGGTTCAC-3′) plus 5B-R for RPL5B.

• Supplemental Figure S4 -

  Fig. S4. The leaf epidermal cell patterns of wild-type and mutant plants. The abaxial leaf surface of Col (A), as2-1 (C), stv1-1 (E), rpl5b (G), ae6-2 (I) and ae5-1 (K). The adaxial leaf surface of Col (B), as2-1 (D), stv1-1 (F), rpl5b (H), ae6-2 (J) and ae5-1 (L). Note that the epidermal cell patterns in the single mutants were all normal. (M-Q) The abaxial epidermal cells of ett-3 arf4-2 (M), ae5-1 ett-3 arf4-2 (N), ae6-2 ett-3 arf4-2 (O), kan1-2 kan2-1 (P), ae5-1 kan1-2 kan2-1 (Q). Note that not only ae5-1 ett-3 arf4-2, ae6-2 ett-3 arf4-2 (data not shown) and ae5-1 kan1-2 kan2-1 produced dramatically reduced numbers of ectopic outgrowths on the leaf abaxial side as compared with those in ett-3 arf4-2 and kan1-2 kan2-1 (see Fig. 7), but the outgrowths were much less pronounced in the triple mutants than those in their corresponding double mutants. ab, leaf abaxial side; ad, leaf adaxial side. Scale bars: 50 µm.

• Supplemental Figure S5 -

  Fig. S5. In situ hybridization using serial sections to analyze FIL and REV expression patterns in ae5-1, as2-101 and as2-101 ae5-1 mutants. Three transverse sections were selected from the same seedling sample of wild-type or mutants. (A-D) The expression pattern of FIL in Ler (A1-3), ae5-1 (B1-3), as2-101 (C1-3) and as2-101 ae5-1 (D1-3). (E-H) The expression pattern of REV in Ler (E1-3), ae5-1 (F1-3), as2-101 (G1-3) and ae5-1 as2-101 (H1-3). The images in the black box are those shown in Fig. 5.

• Supplemental Figure S6 -

  Fig. S6. ae6-2 (rpl5a) rpl5b double mutations may result in embryonic lethality. (A) Genotyping of F2 plants of a cross between ae6-2 and rpl5b. The primers used in genotyping are shown in the upper panels. Note that DNA bands with different sizes could be well separated, and the electrophoresis results indicated that plants with different genotypes in the F2 progeny could be clearly distinguished using these PCR primers. The F2 plants could be grouped into only two categories: one showed the wild-type phenotypes and the other exhibited the similar ae6-2 or rpl5b phenotypes. We analyzed a total of 92 such F2 plants, including 32 wild-type-like plants and 60 ae6-2- or rpl5b-like plants. To our surprise, we only identified the wild-type, ae6-2/+ and rpl5b/+ plants in the wild-type-like progeny, and ae6-2, rpl5b and ae6-2+ rpl5b/+ plants in the ae6- or rpl5b-like progeny, but did not find any plant with the ae6-2 rpl5b, ae6-2 rpl5b/+ or ae6-2/+ rpl5b genotype. Our other results suggest that mutations ae6-2 rpl5b, ae6-2 rpl5b/+ and ae6-2/+ rpl5b may result in embryonic lethality (see below). (B) A Col seedling. (C) An F1 seedling of a cross between ae6-2 and rpl5b. Note that the ae6-2/+ rpl5b/+ plant exhibited pale-green leaves, similar to those of its parents. (D) Silique phenotypes of wild-type Col, ae6-2, rpl5b and ae6-2/+ rpl5b/+. Note that ae6-2/+ rpl5b/+ siliques were slightly shorter than those of the wild type and single-mutant plants. (E) In a segregating F2 population, ae6-2/+ rpl5b/+ plants produced siliques containing many aborted seeds (asterisks), whereas its sibling plants including Col, rpl5a and rpl5b, which were grown in the same conditions as the rpl5a/+ rpl5b/+ plants, formed normal seeds. Scale bar: 0.5 cm in B,C; 1 cm in D; 1 mm in E.