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First published online 19 May 2004
doi: 10.1242/dev.01168

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Identification of new members of Fertilisation Independent Seed Polycomb Group pathway involved in the control of seed development in Arabidopsis thaliana

¹ EMBO YIP Team, Unité Mixte de Recherche 5667, IFR128 BioSciences Lyon-Gerland, Ecole Normale Supérieure de Lyon, 46 allée d'Italie, F-69364 Lyon cedex 07, France
² Institute of Plant Biology and Zürich-Basel Plant Science Center, University of Zürich, Zollikerstrasse 107, CH-8008 Zürich, Switzerland

View larger version (121K): [in a new window]   Fig. 1. Ontogeny of the endosperm posterior pole. 3D reconstruction of the endosperm posterior pole. Inset shows the location of the posterior pole in a schematic whole seed. Nuclei in the cyst have been labelled in red and other nuclei in yellow. The anteroposterior (AP) axis is indicated by the green double arrow. (A) Embryo dermatogen stage, endosperm stage VII with approx. 50 nuclei. Two large nuclei in the cyst have undergone two cycles of endoreduplication and are embedded in a pool of cytoplasm. Other nuclei in the peripheral domain are surrounded by a small mass of cytoplasm that constitutes a NCD. Scale bar: 25 µm. The 3D reconstruction was obtained from 85 optical sections of 0.4 µm thickness. (B) Early globular stage embryo, endosperm stage VIIIa with approx. 100 nuclei. Above the cyst that still contains only two large nuclei, are two masses of cytoplasm that each contain two nuclei (arrows). Other NCDs are still attached to the walls of the peripheral endosperm. Scale bar: 25 µm. The 3D reconstruction was obtained from 46 optical sections of 0.65 µm thickness. (C) Early heart stage embryo, endosperm stage IX with cellularisation initiated in the micropylar endosperm at the anterior pole (not visible). The 3D reconstruction is represented as an open book split in the middle, along a plane aligned with the anteroposterior axis. The cyst contains multiple nuclei of similar size to those in NCDs. Above the cyst, are observed seven nodules that contain from two up to ten nuclei. In more anterior domains, free NCDs line the wall of the peripheral endosperm. Scale bar: 50 µm. The 3D reconstruction was obtained from 96 optical sections of 0.4 µm thickness.

View larger version (91K): [in a new window]   Fig. 2. Live imaging of migration and fusions of NCDs at the endosperm posterior pole. (A) Confocal sections selected from a time-lapse series of 67 images acquired every 10 minutes. The whole video can be seen at http://dev.biologists.org/supplemental/. The endosperm endoplasmic reticulum is labelled with mGFP5 expressed under the control of the specific enhancer KS22 (Boisnard-Lorig et al., 2001). Hence each NCD is labelled individually and the cyst (c) appears as a large fluorescent mass at the posterior pole. The seventh mitotic division has taken place 40 minutes before time 0 minute. After mitosis, NCDs migrate toward the cyst (170 minutes). Later, a NCD migrates towards a neighbouring NCDs closer to the posterior pole and eventually fuses with it (370-430 minutes). The resulting nodule attracts a third NCD while other NCDs migrate and fuse (590-660 minutes). (B) A time-lapse series similar to the one shown in A, selected from 82 images acquired every 10 minutes, showing the absence of oriented migration and non-specific fusions of NCDs in the mutant fis2-3 background. The seed was selected as fis2-3 at the mid-globular stage on the basis of its strong fluorescence, persistent in all domains of the endosperm, provided by the activity of the enhancer KS117 that drives expression of mGFP5 (Sørensen et al., 2001). A mitotic division has taken place 20 minutes before time 0 minute and no posterior migration of NCDs is observed in comparison to WT. No specific migration nor oriented fusion is observed later in development and unusually large nodules assemble as a result of `passive' engulfing by growing cytoplasm (360-510 minutes). Gradually, the polarised anteroposterior arrangement of NCDs, nodules and cyst is lost (810 minutes). Scale bars: 20 µm (all sections).

View larger version (139K): [in a new window]   Fig. 3. Comparison of the organisation of the endosperm posterior pole in the WT and in fis2-3. (A) Confocal section of the endosperm posterior pole from the abaxial side of a WT seed at the torpedo stage. The endosperm is cellularised with the exception of the posterior pole that consists of the cyst containing nuclei with very dense chromatin. (B) In contrast to WT, fis2 endosperm is not cellularised at the torpedo stage and its posterior pole consists of a large cyst (c) and multiple large multinucleate nodules (n). Nuclei in the cyst and in nodules show a chromatin organisation similar to nuclei in the peripheral endosperm. Scale bars: 20 µm.

View larger version (89K): [in a new window]   Fig. 4. Gametophytic maternal effect mutants with a fis phenotype. KS117 GFP is misexpressed. In mutant seeds, the level of GFP accumulation is much higher and the expression is not restricted to the posterior pole in mutant seeds. More than 25% of seeds abort. One line from each mutant group is presented here. (A-G) Seeds observed by epifluorescence stereomicroscopy. (H-N) Seeds observed by light microscopy. All aborting seeds are indicated by red dots. (A,H) WT KS117; (B,I) dme-4/DME; (C,J) mea-6/MEA; (D,K) fis2-6/FIS2; (E,L) fie-11/FIE; (F,M) bga-1/BGA; (G,N) msi1-2/MSI1. Scale bar: 200 µm (all images).

View larger version (134K): [in a new window]   Fig. 5. Microscopic analysis of phenotypes in bga/BGA seeds. (A,B) WT embryos at mid globular and mid heart stage, respectively. (C) Cellularised endosperm in a WT seed at mid heart stage. (D,E) bga/BGA embryos in the same siliques as those in A and B, respectively. (F) Endosperm is not cellularised in a bga/BGA seed at the same stage as in C. Scale bars: 30 µm.

View larger version (88K): [in a new window]   Fig. 6. Confocal sections of bga-1 and msi1-2 mutant seeds. (A) In WT seeds at early torpedo stage (emb), peripheral endosperm is cellularised (cpe). The cyst (cy) and small nodules (no) are visible at the posterior pole. (B) In siliques at the same stage, bga seeds show a delay in embryo development, although morphology is normal. Peripheral endosperm is not cellularised and the posterior pole overproliferates. (C) msi1-2 seeds have arrested embryos. Peripheral endosperm is not cellularised and the posterior pole strongly overproliferates. Scale bar: 100 µm (A-C).

View larger version (148K): [in a new window]   Fig. 7. Microscopic analysis of msi1-2/MSI1 and msi1-2/msi1-2 seeds. (A,B) WT embryo at dermatogen and mid heart stage, respectively. em: embryo proper, su: suspensor. (C) Cellularised endosperm in a WT seed at the same stage as in B. (D,E) msi1-2/MSI1 embryos in same siliques as A and B, respectively. (F) Endosperm is not cellularised in a msi1-2/MSI1 seed at the same stage as in C. (G,H) msi1-2/msi1-2 embryos in same siliques as A and B, respectively. (I) Endosperm is not cellularised in a msi1-2/msi1-2 seed at the same stage as in C. Nucleoli are variable in size. Scale bars: 50 µm.

View larger version (145K): [in a new window]   Fig. 8. Autonomous seed development in gametophytic maternal effect mutants. Mature flower buds were emasculated. (A-G) After 7 days, pistil elongation was scored. Scale bar: 1 mm. (H-N) Pistils were then slit open in order to observe seeds (arrows) and undeveloped ovules. Scale bar: 200 µm. (A,H) WT KS117; (B,I) dme-4/DME; (C,J) mea-6/MEA; (D,K) fis2-6/FIS2; (E,L) fie-11/FIE; (F,M) bga-1/BGA; (G,N) msi1-2/MSI1.

View larger version (64K): [in a new window]   Fig. 9. KS117 GFP marker expression 9 days after pollination of heterozygous gametophytic maternal mutants by a demethylated genome. (A) mea-6/MEA x MET1 a/s; (B) fis2-6/FIS2 x MET1 a/s; (C) fie-11/FIE x MET1 a/s; (D) dme-4/DME x MET1 a/s; (E) bga-1/BGA x MET1 a/s; (F) msi1-2/MSI1 x MET1 a/s. Two types of seeds are produced when fis/FIS mutants are fertilised by demethylated MET1 a/s pollen: the small seeds (arrows) result from WT ovule development, the large seeds (arrowheads) from mutant ovules. GFP expression is low and restricted to the posterior pole in small seeds. In mea/MEA (A), fis2/FIS2 (B), dme/DME (D) and bga/BGA (E) seeds, KS117 GFP expression is properly restricted to the cyst, even when this structure is larger than in WT (A). In contrast, in fie/FIE (C) and msi1/MSI1 (F) seeds, KS117 GFP is over-expressed and not restricted to the posterior cyst (arrowheads), as when pollinated by WT. Scale bar: 200 µm (all images).

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