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doi: 10.1242/10.1242/dev.00458

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The Arabidopsis mutant feronia disrupts the female gametophytic control of pollen tube reception

Norbert Huck1, James M. Moore1,2,3, Michael Federer1 and Ueli Grossniklaus1,2,*

1 Institute of Plant Biology & Zürich-Basel Plant Science Center, University of Zürich, Zollikerstrasse107, 8008 Zürich, Switzerland
2 Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
3 Graduate Program in Genetics, State University of New York, Stony Brook, NY 11794, USA



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  		Fig. 1. (A) Schematic drawing of a mature female gametophyte (FG) enclosed within the ovule. The FG consists of the two synergid cells (sc), an egg cell (ec), the central cell (cc) and three antipodal cells (ac). (B) Path of the pollen tube (PT) through the style and transmitting tissue towards the ovules. (C) PT entering the FG through the receptive synergid.

 


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  		Fig. 2. The feronia mutant is semisterile and shows reduced transmission through the female gametophyte. (A) Wild-type siliques show full seed set. (B) In feronia siliques only about half of the ovules develop into seeds, the others remain unfertilized (arrows). (C) Southern blot using Ds 5' end-specific probe indicates a single Ds insertion in the feronia mutant. The observed fragment sizes are consistent with the expected sizes after restriction with EcoRI (3.2 kb) and HindIII (2.0 kb). (D) Transmission efficiency (TE) of the kanamycin resistance gene, which is tightly linked to feronia, through wild-type and feronia gametophytes [TE=(kanamycin resistant seedlings)/(kanamycin sensitive seedlings); progeny of reciprocal out-crosses to the wild type]. In feronia the transmission through the male gametophyte is reduced to 78.5% and through the female gametophyte to 14.5%.

 


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  		Fig. 3. Invading PT phenotypes in the feronia mutant. (A-C,H,J) Cleared whole-mount preparations. (D,E,I) Ovules stained with Aniline Blue to visualize the PT. (F,G) Crosses to PT marker line expressing the ß-glucuronidase (GUS) gene. (A) Mature FGs of wild-type and feronia (fer/+) plants before fertilization are indistinguishable from each other and contain synergid cells (sc), egg cell (ec) and central cell (cc). (B) Fertilized wild-type ovules at 24 HAP that contain an elongated zygote (arrow) and free nuclear endosperm (arrowheads). (C) Unfertilized feronia (fer/+) ovule at 24 HAP. The secondary endosperm nucleus (sen) of the central cell and the egg cell nucleus (ecn) are visible. The pollen tube (arrow) enters the ovule but forms an entangled structure (arrowhead) within the FG. (D) Wild-type ovule after fertilization. The PT (arrow) terminated in the micropylar area (mi) of the embryo sac. (E) feronia mutant in which the PT invades the micropylar area (arrow). (F) In wild-type ovules the GUS-activity of the PT remains restricted to the micropylar area of the FG (arrow). (G) In mutant FG the GUS signal is observed in the entire micropylar part of the FG. Owing to the strong GUS activity, the stain diffused into the central cell. (H,I) The PT enters the central cell (arrowheads) after coiling in the micropylar area (arrow). (J) Formation of free nuclear endosperm (arrowheads) after the PT has invaded the FG (arrow). Scale bars: 30 µm.

 


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  		Fig. 4. Supernumerary PTs enter mutant feronia FGs. (A,D-F) Cleared whole-mount preparations. (B) Ovule stained with Aniline Blue to visualize the PT. (C) Confocal micrograph with transections in z-axis. A and B show the same ovule in the same focal planes. At least two PTs (arrows, PT1 false coloured in red, PT2 in green) enter the ovule at the micropyle (arrowhead). (C) Two pollen tubes (pt1, pt2) enter the ovule and grow through the micropyle towards the embryo sac. The top and the side panel show the transections of the confocal image stack in z-axis along the lines shown in the main panel. Arrows in the transections mark the two PTs reaching the FG. (D,E) Successive arrival of PTs at ovules at 12 HAP. (D) The first PT (arrow, false coloured in green) has already entered the micropyle. (E) Different focal plane of the same ovule. The second PT approaches the micropyle (arrowhead, false coloured in red). The arrow indicates the first PT entering the ovule. (F) Two PTs (arrows) have invaded (arrowhead) and entered the FG and proceeded into the central cell (cc). This specimen was derived from a tetraploid feronia plant. Scale bars: 30 µm.

 


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  		Fig. 5. Synergid-specific GUS activity of one out of three enhancer detector lines in wild-type and feronia ovules. (A) In mature wild-type ovules before fertilization, the GUS expression is restricted to the synergid cells (sc). (B) The GUS activity decreases significantly in wild-type ovules after fertilization (arrow). (C) In feronia (fer/+) ovules the synergid cells (sc) are stained before fertilization. (D) After PT invasion the GUS activity persists in the synergids (arrow) of mutant feronia embryo sacs. The invading PT appears to be covered by the GUS stain, indicating that it coils within the synergid. Scale bars: 30 µm.

 


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  		Fig. 6. Cytological analysis of pollen tube entry into feronia embryo sacs. (A,B) Semi-thin sections stained with Toluidine Blue. (C-F) Transmission electron micrographs of invaded feronia FG at 24 HAP. (A) The winding PT (pt) is covered by the dark staining remains of the synergid cell (sc). (B) PT invasion in mutant ovules is in most cases restricted to the synergid and does not enter the central cell (cc). (C) The PT enters the synergid and bifurcates (arrow) immediately after entry into the degenerated synergid (dsy). The egg cell membrane (arrowheads) appears to be intact and the secondary endosperm nucleus (sen) is present. (D) Adjacent but not successive section of the same sample as shown in C at higher magnification. The PT passes the filiform apparatus (fa) and enters the FG. Remains of the degenerated synergid surround the entering PT. (E) Branching (arrow) of the PT within the synergid. (F) The invading PT rearranges the micropylar area of the embryo sac, but the egg cell membrane appears to be intact (arrowheads). Scale bars: (A,B) 25 µm; (C-F) 5 µm.

 




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