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Roles of PIN-FORMED1 and MONOPTEROS in pattern formation of the apical region of the Arabidopsis embryo

Mitsuhiro Aida^1,*,, Teva Vernoux^2,, Masahiko Furutani¹, Jan Traas^2, and Masao Tasaka^1,

¹ Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara 630-0101, Japan
² INRA, Laboratoire de Biologie Cellulaire, Route de Saint Cyr, 78026 Versailles cedex, France
^* Present address: Department of Molecular Cell Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands
These authors contributed equally to the research

View larger version (84K): [in a new window]   Fig. 1. Phenotypes of wild type, cuc1 cuc2, pin1 and pin1 cuc1 cuc2. (A-H) 4-day-old seedlings of wild type (A,B), cuc1 cuc2 (C,D), pin1-3 (E,F) and pin1-3 cuc1 cuc2 (G,H). In B,D and H, seedlings were cleared to visualize the vascular pattern. A wild-type seedling has two symmetrically arranged, completely separated cotyledons (A) and each cotyledon contains a single mid-vein (B, arrows). cuc1 cuc2 has two bilaterally symmetrical cotyledons, as revealed by two splits at the top (arrowheads in C,D) and a vascular pattern similar to wild type (D). Arrow in D indicates one of the two mid veins (the other one is out of focus). pin1 shows variable phenotypes including two completely separated cotyledons (E), partial fusion and increased number of cotyledons (F, left and center) and a wide collar-shaped cotyledon (F, right). The fused part is indicated by arrowheads. pin1 cuc1 cuc2 shows a radially symmetrical morphology as revealed by complete cotyledon fusion (G) and evenly distributed vascular bundles (H). (I-L) Scanning electron micrograph images of wild-type (I), cuc1 cuc2 (J), pin1-3 (K) and pin1-3 cuc1 cuc2 (L) embryos. Note that two splits are apparent at the top of a cup-shaped cotyledon in cuc1 cuc2 (J, arrowheads) while no such split is found in pin1 cuc1 cuc2 (L).

View larger version (105K): [in a new window]   Fig. 2. Expression of PIN1 during early embryogenesis in cuc1 cuc2 double mutants. PIN1 mRNA was detected using in situ hybridization. (A-D) Expression of PIN1 in wild-type embryos. (E,F) Expression of PIN1 in cuc1 cuc2 embryos. Arrowheads in A indicate the future sites of cotyledon emergence. Note the similar expression pattern of PIN1 in wild-type and cuc1 cuc2 embryos. Scale bar, 40 µm.

View larger version (65K): [in a new window]   Fig. 3. Expression of CUC1 and CUC2 in pin1 embryos. CUC1 and CUC2 mRNA was detected using in situ hybridization. (A,B) Schematic representation of wild-type expression patterns of CUC1 (A) and CUC2 (B) in a median sagittal section. Relative intensities of the signal are represented by dark (strong) and light (weak) blue. (C,E) CUC1 expression in wild type (C) and pin1-3 (E) in serial longitudinal sections. (D,F) CUC2 expression in wild type (D) and pin1-3 (F) in serial longitudinal sections. Arrow in F indicates an example of a weak spot of signal, which is found at the side of the embryo in a few cases. Scale bar, 50 µm for C-F.

View larger version (124K): [in a new window]   Fig. 4. Double mutant phenotypes of pin1 cuc1. (A, left and center) 4-day-old seedlings of pin1-3 cuc1 with significant splits at the top (left) and no splits (center) and (right) 6-day-old seedling of pin1-6 cuc1. (B-D) Histological sections of the SAM stained with Toluidine Blue. (B) Wild type. (C) pin1-6. (D) pin1-6 cuc1. Scale bars, 50 µm. (E-G) Confocal images of the apices of mature embryos stained with propidium iodide. (E) Wild type. (F) pin1-3. (G) pin1-3 cuc1. The SAM is the area of small densely stained cells. Note the SAM is smaller in pin1-3 cuc1 embryo. Scale bars, 50 µm.

View larger version (93K): [in a new window]   Fig. 5. Phenotype of pin1 stm double mutants. (A-D) 7-day-old seedlings. (A) Wild type. (B) pin1-6 single mutant. (C) stm single mutant. (D) pin1-6 stm double mutant. (E-H) Seedlings were cleared to visualize the vascular pattern. (E) Wild type. (F) pin1-6 single mutant. (G) stm single mutant. (H) pin1-6 stm double mutant. The vasculature shows a bilateral symmetry in wild type and stm while it is asymmetric in pin1-6. In the double mutant the vasculature shows a radial symmetry. (I-L) Histological sections of the SAM stained with Toluidine Blue. (I) Wild type. (J) pin1-6. (K) stm. (L) pin1-6 stm. Scale bars, 50 µm.

View larger version (98K): [in a new window]   Fig. 6. Relationship between CUC and MP genes. (A) mp seedlings with completely separated (left) and variably fused (middle and right) cotyledons. Note that the size of the cotyledons is unequal in the middle seedling. (B,C) Expression patterns of CUC1 (B) and CUC2 (C) in serial longitudinal sections of mp embryos. CUC1 and CUC2 mRNA was detected using in situ hybridization. Scale bar, 50 µm for B and C. (D) mp cuc1 seedlings. The seedling at the left has cotyledons fused at one side while the seedling at the right has a ‘cup-shaped’ cotyledon. (E) mp cuc2 seedling with cotyledons fused at one side.

View larger version (27K): [in a new window]   Fig. 7. Regulation of expression patterns of CUC1 and CUC2 at the early heart stage. PIN1 and MP repress CUC1 expression in the cotyledons and promote CUC2 in the cotyledon boundaries.

View larger version (22K): [in a new window]   Fig. 8. A model for the patterning of the apical region of the embryo. Solid arrows indicate activation. Dotted arrows indicate an effect on spatial expression. PIN1 promotes auxin transport and creates a specific auxin distribution. The auxin distribution activates the expression of CUC2 and influences the spatial expression pattern of CUC1. MP regulates CUC gene expression in response to auxin signals. MP could also contribute to auxin distribution through promoting auxin transport. CUC1 and CUC2 redundantly promote cotyledon separation as well as expression of STM, the latter of which in turn promote SAM formation. STM also contributes to cotyledon separation through regulating spatial expression of CUC1 and CUC2 at the late embryo. In addition, STM promotes the CUC1 activity from early embryogenesis on.