1. Volume 13, Issue 5, Pages 432-436 (March 2003)
Female Control of Male Gamete Delivery during Fertilization in Arabidopsis thaliana 
Nicolas Rotman, Frédérique Rozier, Leonor Boavida, Christian Dumas, Frédéric Berger, Jean-Emmanuel Faure 
Current Biology 
Volume 13, Issue 5, Pages (March 2003)
DOI: /S (03)

2. Figure 1
Embryo Sac Structure, as Observed with Normarski Microscopy after Clearing of A. thaliana Ovules
(A) Unfertilized embryo sac in a wild-type pistil at anthesis. It contains a central cell with a single nucleus (nucleolus: ccn), and a highly polarized egg cell (nucleolus: ecn). One of the two synergid cells is visible (nucleolus: syn), with a characteristic polarity opposite to that of the egg cell. “ot” indicates the outer integuments of the ovule.
(B) Unfertilized embryo sac in a srn/SRN pistil 2 days after pollination. Tangled material (tm) is visible in the micropylar part. There is no sign of fertilization; we can see the egg cell nucleus (nucleolus: ecn) as well as the central cell nucleus (nucleolus: ccn).
(C) Wild-type-looking ovules in a srn/SRN pistil at 2 days after pollination are fertilized: The embryo proper is at a two-cell stage (emb), and the endosperm (en) developed into a syncytial structure containing several nuclei (nucleoli: arrow-heads). The scale bar represents 10 μm.
Current Biology  , DOI: ( /S (03) )

3. Figure 2 Pollen Tube Penetration into Wild-Type and srn Embryo Sacs
Micropylar ends of embryo sacs fixed at 7 hr after pollination and observed with CLSM.
(A) Unfertilized embryo sac in a srn/SRN pistil. Projection of three optical sections (1.4 μm thick each) from the same embryo sac. A single nucleus with its nucleolus (ccn) is visible in the central cell. The egg cell is highly polarized and has its nucleus and nucleolus (ecn) located next to the central cell. The two synergid cells are inversely polarized, with their nuclei and nucleoli (syn) close to the filiform apparatus (fa).
(B) Optical section (1.2 μm) in a sirène embryo sac penetrated by a pollen tube (pt). The pollen tube grows deep into the embryo sac toward the central cell.
(C) Projection of selected elements from seven optical sections (1.2 μm each) in the same sirène embryo sac as in (B). The tube (colorized in green) reaches the central cell (nucleolus in yellow), whereas the two synergid cells (nucleoli in blue) and the egg cells are intact (nucleolus in red).
(D) Projection of six optical sections (1.6 μm each) from a wild-type embryo sac penetrated by a pollen tube, with a degenerated synergid cell (dsy). The scale bar represents 10 μm.
Current Biology  , DOI: ( /S (03) )

4. Figure 3
sirène Affects the Pollen Tube Morphology within the Embryo Sac
(A–C) The micropylar ends of ovules from wild-type (A) and srn/SRN pistils (B and C) were collected at anthesis and stained with aniline blue. (A) In wild-type pistils, the pollen tube (pt) penetrates the ovule micropyle (m) and reaches the embryo sac (es). (B) By contrast, the pollen tube penetrates the ovule micropyle (m) but forms a tangled structure into the sirène embryo sac; we interpret this structure as a pollen tube overgrowth (pto). “ot” indicates the outer integuments of the ovule. (C) Situation in which two pollen tubes (pt1 and pt2) have penetrated the micropyle of an ovule and formed a coiled structure into a sirène embryo sac.
(D) Interpretative scheme for the sirène phenotype. The pollen tube has entered the embryo sac and coiled up in the micropylar part, whereas the egg cell (nucleus: ecn) and the central cell (nucleus: ccn) are left unfertilized. Abbreviations are as follows: fa, filiform apparatus; and f, funiculus. The scale bar represents 10 μm.
Current Biology  , DOI: ( /S (03) )

5. Figure 4
Time-Lapse Imaging of Pollen Tube Penetration into the Embryo Sac
(A) The pistil (p) to be imaged is mounted on a slide (s) under a coverslip (c), with the floral pedonculus dipping in the MS medium (m) itself surrounded by high vacuum grease (g) to seal the imaging chamber.
(B) Two rows of ovules are exposed to imaging after an incision is made along the pistil, from pedonculus (fp) to stigma (st).
(C) Ovules (ov) within an open half are visible with their funiculus (f) and micropyle (m). Enlargement of the red box in panel (B).
(D–E) Time-lapse series of pollen tube penetration into wild-type (D) and srn (E) embryo sacs, 6 to 8 hr respectively after in planta pollination. Growing pollen tubes are imaged with confocal laser scanning microscopy. Fluorescence from EGFP expressed in pollen tubes is shown in green, and autofluorescence in the ovules and embryo sacs is shown in red. A first pollen tube (pt1) has penetrated the micropyle (m) of the ovule (ov) and grows into the embryo sac, and a second one (pt2) stops growth on the funiculus (f). Cytoplasm and organelles in the central cell are brightly autofluorescent (cc). (D) In the wild-type situation, the pollen tube forms an extension (white arrowhead on the 4 min panel) within the embryo sac, toward the central cell, and releases its contents in an explosive manner (7 min panel). The panels showing from 4 min to 10 min correspond to enlargements of the yellow box in the 0 min panel (E). Instead, in a sirène embryo sac the pollen tube coils up within the space between the micropylar end and the central cell. The pollen tube contents are not released during the imaging time. Panels showing from 76 min to 154 min correspond to enlargements of the yellow box in the 0 min panel. The scale bar represents 500 μm in panels (A)–(C) and 10 μm in panels (D) and (E).
Current Biology  , DOI: ( /S (03) )