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doi: 10.1242/10.1242/dev.00292

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Regulation of shoot epidermal cell differentiation by a pair of homeodomain proteins in Arabidopsis

Mitsutomo Abe^1,*, Hiroshi Katsumata¹, Yoshibumi Komeda^1,2 and Taku Takahashi^1,

¹ Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan
² Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Tokyo 113-0033, Japan
^* Present address: Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan

View larger version (77K): [in a new window]   Fig. 1. Structure and deduced amino acid sequence of PDF2. (A) Genomic structure of PDF2 and ATML1. Black boxes represent exons. Start ATG and termination codons are indicated. T-DNA insertion sites in pdf2-1 and atml1-1 are shown. The upstream regions of both genes contain L1 box sequences (gray boxes) associated with putative WUS target sites (underlined). (B) The predicted amino acid sequence of the PDF2 protein. The boxed area indicates a homeodomain. Amino acids forming a ZIP motif are shaded. A START domain is underlined.

View larger version (87K): [in a new window]   Fig. 2. Expression pattern of PDF2. (A) RNA gel blot analysis of PDF2 mRNA. Total RNA was isolated from whole seedlings (Se), roots (R), leaves (L), stems (St), flower-bud clusters (F) and siliques (Si). Seedlings were grown for 10 days in MS plates. The EF1 probe was used as a control for loading and transfer. (B-G) In situ localization of PDF2 mRNA. Longitudinal sections of a vegetative shoot meristem of a 10-day-old seedling (B), apical inflorescence and floral meristems (C), a flower bud (D), ovule primordia (E), a quadrant-stage embryo (F) and an early globular-stage embryo (G) were hybridized with the antisense PDF2 RNA probe.

View larger version (86K): [in a new window]   Fig. 3. Interaction of the PDF2 gene product with the L1 box in vitro. Gel retardation assays were performed using a labeled 21-bp L1 box probe (L1) derived from the PDF1 promoter, together with the MBP alone or the PDF2 fusion protein (MBP-PDF2) as indicated. The wedge indicates increasing amounts of 100-, 300- and 1000-fold molar excesses of unlabeled L1 probe DNA in competition assays. An assay using a mutated probe (mL1) with MBP-PDF2 is also shown.

View larger version (104K): [in a new window]   Fig. 4. Phenotype of the atml1-1 pdf2-1 mature embryo. (A) Wild-type (WT) and double mutant mature embryos dissected from dry seed. Samples were cleared and examined under Nomarski optics. Scale bar: 100µm. (B) SEM view of the double mutant mature embryo. Scale bar: 100 µm. (C,D) Median sections through the double mutant shoot apex (C) and the double mutant root apex (D). Scale bars: 50 µm.

View larger version (90K): [in a new window]   Fig. 5. Post-embryonic growth in atml1-1 pdf2-1. (A) Ten-day-old wild-type seedling. (B) Ten-day-old seedling of the double mutant. (C) Eighteen-day-old seedling of the double mutant. (D) SEM view of the double mutant leaf. Scale bar: 0.5 mm. (E-H) SEM micrographs showing the adaxial (E,F) and abaxial (G,H) surfaces of leaves of the wild type (E,G) and the double mutant (F,H). No epidermal cell layer is present in the double mutant (F,H). Wild-type leaves were partially peeled to show the mesophyll underneath the epidermis (E,G). Scale bars: 50 µm. (I) Cross-section of a double mutant leaf. Scale bar: 0.5 mm. (J) A cluster of stomatal guard cells found in a double mutant leaf. Scale bar: 10 µm.

View larger version (80K): [in a new window]   Fig. 6. Effect of atml1-1 pdf2-1 on the expression of L1 layer-specific genes. RT-PCR analysis of the expression of PDF1, PDF2, ATML1 and ACR4 is shown. Total RNA was prepared from aerial tissues of 10-day-old mutant and wild-type seedlings. Expression of STM was also examined as a control.

View larger version (92K): [in a new window]   Fig. 7. Northern analysis of mRNA isolated from flower-bud clusters of wild-type and transgenic plants with the 35S::PDF2 construct. Lines 1, 10 and 12 represent PDF2 overexpression plant lines, and lines #3 and #5 represent co-suppression plant lines. The relative mRNA levels are corrected with the EF1 mRNA as a control, compared with wild-type signal levels and indicated below the signals.

View larger version (144K): [in a new window]   Fig. 8. Phenotypes of PDF2 overexpression plants. (A) Thirty-day-old wild-type plant. (B) Forty-five-day-old PDF2 overexpression plant (line 1). (C,D) In situ localization of PDF2 mRNA in apical inflorescence meristems of PDF2 overexpression plants (line 1). Longitudinal sections were hybridized with antisense (C) or sense (D) PDF2 RNA probe.

View larger version (126K): [in a new window]   Fig. 9. Phenotypes of PDF2 co-suppression plants. (A) Wild-type flower. (B) Flower of the PDF2 co-suppression plant (line 3). (C,D) SEM views of a stage-nine flower of the wild type (C) and the PDF2 co-suppression plant (D). The adaxial sepal has been removed. (E,F) Longitudinal sections of an apical inflorescence meristem of the wild type (E) and the PDF2 co-suppression plant (F). (G-N) SEM views of epidermal cells of the wild type (G,I,K,M) and PDF2 co-suppression plants (H,J,L,N). Epidermal cells of the adaxial side of stage-nine petals (G,H), the adaxial side of stage-13 petals (I,J), the abaxial side of stage-13 petals (K,L) and the adaxial side of stage-nine sepals (M,N) are shown. Scale bars: 25 µm in E,F; 10 µm in G-L; 50 µm in M,N.

View larger version (34K): [in a new window]   Fig. 10. (A) Schematic summary of regulatory functions of PDF2 (blue) and ATML1 (red) in L1 layer-specific gene expression. The illustration incorporates potential formation of homo- and heterodimers. Red arrows indicate transcriptional activation mediated by the interaction of PDF2 and/or ATML1 with the L1 box located upstream of each gene. (B) A hypothetical model in which the L1 layer is established and maintained in the embryo. Continuation of an autoregulatory loop for the PDF2 and ATML1 expressions is maintained in the protodermal layer, and an as-yet-unidentified suppressor(s) functions (orange) in switching off the loop in the basal and inner cells of the 16-cell embryo.