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First published online 9 April 2008
doi: 10.1242/dev.014993

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The Arabidopsis OBERON1 and OBERON2 genes encode plant homeodomain finger proteins and are required for apical meristem maintenance

Shunsuke Saiga¹, Chihiro Furumizu¹, Ryusuke Yokoyama^2,*, Tetsuya Kurata^2,, Shusei Sato³, Tomohiko Kato^3,, Satoshi Tabata³, Mitsuhiro Suzuki¹ and Yoshibumi Komeda^1,

¹ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Tokyo 113-0033, Japan.
² Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan.
³ Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan.

View larger version (49K): [in this window] [in a new window]   Fig. 1. Deduced amino acid sequences of OBE1 and OBE2, and subcellular localization of the OBE1 protein. (A) Alignment of the predicted amino acid sequences of OBE1 and OBE2. Identical amino acid residues and similar amino acid residues are highlighted by black and gray backgrounds, respectively. The PHD finger domain and coiled-coil domain are underlined with blue and black, respectively. (B) Genomic structure of OBE1 and OBE2. Black boxes represent exons. Positions of the start and termination codons are indicated. T-DNA insertion sites in obe1-1 and obe2-1, and the location of obe2-2 mutation are shown. Arrows represent the positions of the primers used to amplify the OBE1 and OBE2 transcripts. (C,D) Confocal images of root hair cells from 35S::OBE1-GFP (C) and 35S::GFP (D) transgenic plants. (C) The OBE1-GFP fusion protein was localized in the nucleus. (D) The GFP signal was detected throughout the cell.

View larger version (101K): [in this window] [in a new window]   Fig. 2. Expression of OBE1 and OBE2. (A) RT-PCR analysis of OBE1 and OBE2 expression in various tissues as shown. Accumulation of EF1 was used as a control. (B-E) Wild-type embryo specimens of in situ hybridization with OBE1 (B,D) and OBE2 (C) antisense probes and (E) OBE1 sense probe. (B) Four-cell stage embryo. (C) Eight-cell stage embryo. Hybridization signals were detected in the embryo proper but not in the suspensor. (D) OBE1 is expressed in all cells of the heart stage embryo. (E) Control hybridization with an OBE1 sense probe at the heart stage. (F) Expression of the OBE1-GFP fusion protein in the torpedo stage embryo from an OBE1p::OBE1-GFP transgenic plant. Scale bars: 25 µm.

View larger version (70K): [in this window] [in a new window]   Fig. 3. Phenotypes of obe1 obe2 mutants. (A-C) Three-day-old seedlings of wild type (A) and obe1 obe2 (B,C). (D-F) Fourteen-day-old seedlings of wild type (D) and obe1 obe2 (E,F). (G-J) Scanning electron micrographs of the shoot apical meristem of 7-day-old wild-type (G), 5-day-old obe1 obe2 (H,I), and 14-day-old obe1 obe2 seedlings (J). (K,L) Histological sections of the shoot apices from 4-day-old seedlings of wild type (K) and obe1 obe2 (L). Scale bars: 1 mm in A-F; 100 µm in G-L.

View larger version (79K): [in this window] [in a new window]   Fig. 4. Root phenotypes of obe1 obe2 seedlings. (A) Three-day-old seedlings of wild type (left) and obe1 obe2 (right). (B,C,E,F) Toluidine Blue staining of transverse (B,E) and longitudinal (C,F) sections of wild-type (B,C) and obe1 obe2 (E,F) roots. (D,G) Visualization of starch granules in the central root cap of wild type (D) or obe1 obe2 (G). Scale bars: 1 mm in A; 25 µm in B-G.

View larger version (123K): [in this window] [in a new window]   Fig. 5. Embryonic development of obe1 obe2 mutants. (A-N) Embryos of wild-type (A-D,I-K) and obe1 obe2 (E-H,L-N) at the early globular (A,E), transition (B,F), early heart (C,D,G,H), heart (I,L) and bent-cotyledon (J,K,M,N) stages. In B, the arrow indicates the longitudinal division of ground tissue cells. (D,H) Serial optical sections of the same embryos shown in C,G, respectively. In E, arrowhead indicates an aberrant oblique cell division. In H, inset represents magnified view of the boxed area. Arrowheads in the inset indicate the periclinal division of the protodermal cells in the apical region. K and N are higher magnification images of the basal region of embryos from J and M, respectively. h, hypophysis; lsc, lens-shaped cell; cic, columella initial cells; QC, quiescent center. Scale bars: 50 µm in A-J,L,M; 25 µm in K,N.

View larger version (116K): [in this window] [in a new window]   Fig. 6. Expression of the shoot apical meristem marker genes in the obe1 obe2 double mutant. (A,B) CLV3::GUS expression in 4-day-old wild-type (A) and obe1 obe2 (B) seedlings. (C,D) WUS::GUS expression in 4-day-old wild-type (C) and obe1 obe2 (D) seedlings. (E-H) CLV3::GUS expression in wild-type (E,G) and obe1 obe2 (F,H) embryos. Embryos shown are at the heart (E,F) and the torpedo (G,H) stages. (I-P) In situ hybridization with WUS (I-L) and STM (M-P) antisense probes hybridized to embryos from self-fertilized OBE1/obe1 obe2/obe2 plant. Longitudinal sections through embryos at different stages of embryogenesis are shown. (I,J) Heart stage embryos. (K,L,O,P) Bent-cotyledon stage embryos. (M,N) Torpedo stage embryos. (J,L,N,P) obe1 obe2 embryos. (I,K,M,O) Wild-type siblings. In obe1 obe2 embryos, weak WUS expression was detected (J), but WUS expression was undetectable at the bent-cotyledon stage (L). STM expression in obe1 obe2 (N,P) was similar to that in wild-type sibling (M,O). Scale bars: 100 µm in A-D; 25 µm in E-P.

View larger version (60K): [in this window] [in a new window]   Fig. 7. Phenotypes of obe1 obe2 stm and obe1 obe2 wus mutant plants. Fourteen-day-old seedlings of wild type (A), wus-1 (B), obe1 obe2 wus-1 (C), obe1 obe2 (D), stm-1 (E) and obe1 obe2 stm-1 (F). Scale bars: 2 mm.

View larger version (131K): [in this window] [in a new window]   Fig. 8. Expression of the root apical meristem marker genes in the obe1 obe2 double mutant. (A,F) QC46 expression in the central root cap of wild-type (A) and obe1 obe2 (F) seedlings. (B,G) QC46 expression in wild-type (B) and obe1 obe2 (G) heart stage embryos. (C-E,H-R) In situ hybridization with WOX5 (C,D,H,I), MP (E,J), PLT1 (K,L,O,P) and SCR (M,N,Q,R) antisense probes hybridized to embryos from self-fertilized OBE1/obe1 obe2/obe2 plant. Longitudinal sections of in situ hybridization specimens are shown. (C,H) Early globular stage embryos. (D,I,L,P) Late globular stage embryos. (E,J,N,R) Heart stage embryos. (K,O) Eight-cell stage embryos. (M,Q) Globular stage embryos. About one quarter of the embryos from self-fertilized OBE1/obe1 obe2/obe2 plants failed to express WOX5 (H,I), PLT1 (O,P) or SCR (Q,R), while the remaining three-quarters of the embryos exhibited wild-type-like expression of the above genes. By contrast, MP expression was detected in all embryos (J). Scale bars: 25 µm in A-J,L-N,P-R; 10 µm in K,O.

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