Fig 1.
Expression pattern of auxin biosynthetic genes in the developing embryo sac.
A, pYUC2-GFPer expression at FG3 stage. B, pYUC8-GFPer expression in the gametophyte at FG4 stage. C, pTAA1 expression at FG4 stage of gametophyte development. D, Segregation of GFP signal in a line hemizygous for pYUC8::GFPer. From the two ovules shown, only one presents GFP detectable inside the embryo sac (marked as G + S). S, indicates sporophytic signal; G, indicates gametophytic signal. N, nucellus. Arrowheads point at embryo sac nuclei.
Fig 2.
Embryo sac developmental defects in mutants impaired in auxin biosynthesis and import.
A-C, WT; D-F, yuc8/yuc8; G-I, taa1/taa1 tar2/TAR2; J, yuc1/yuc1 yuc2/yuc2; aux1 lax1 lax2; K, aux1 lax1 lax2 lax3. A, WT 4-nucleate embryo sac at FG4 stage. B, WT 8-nucleate embryo sac prior to cellularization. C, WT mature 4-celled embryo sac yuc8 mutant embryo sac at FG7 stage showing defects in polar nucleus migration. D, yuc8 mutant gametophyte at FG4 stage containing only 2 nuclei. A comparable WT gametophyte will typically carry 2 nuclei at this stage. E, yuc8 mutant ovule at FG5 stage with a 2-nucleate arrested embryo sac. F, Mature yuc8mutant embryo sac showing defective polar nucleus migration, no antipodals are visible at this stage. G, A mutant ovule, with only 2 nuclei in the coenocytic embryo sac. H, Mutant ovule with 3-nucleate gametophyte. Unlike clearly polarized nuclei in the WT, these nuclei are scattered in the cytoplasm. I, Mature mutant embryo sac showing defective polar nucleus migration, antipodals are completely degenerated by this stage. J, A yuc1 yuc2 double mutant showing miss-polarized micropylar nuclei, giving rise 2 egg cell-like structures instead of 1. K, Ovule from a triple mutant aux1 lax1 lax2 showing an embryo sac arrested at FG2 stage. The arrows point at the nuclei inside the embryo sac. L, Ovule from a quadruple mutant aux1 lax1 lax2 lax3 showing a collapsing embryo sac containing only two nuclei (arrows). Except for the phenotypes shown in panels D and G, all mutant phenotypes shown are terminal and observed at late stages of ovule development as indicated in Table 1 and in S1 Table. Cv, central vacuole; Ec, egg cell; PN1, polar nucleus 1; PN2, polar nucleus 2; Sc, synergid cell. Scale bar, 50 μM for A-J and 20 μM for K-L.
Fig 3.
Changes in auxin homoeostasis lead to altered cell specification.
A, D, WT. B and C, yuc1/yuc1 yuc2/YUC2. E, yuc8/yuc8.A-C, green signal indicates the expression of central cell specific marker, and red indicates egg cell specific marker. The yellow color observed results from the overlapping of the red colour (egg or egg-like nuclei) on the green background of the central cell in the confocal image. D-E, green signal indicates synergid cell-specific marker, and red indicates egg cell-specific marker. A, WT ovule showing a single egg cell (Red) and a central cell (green) at FG7 stage. B, A mutant ovule at FG7 stage showing 2 egg cells and 1 central cell. C, an FG7 mutant ovule showing 3 egg cells. D, A WT ovule showing normal polarization of synergid nuclei towards the micropylar end. E, merged image of a yuc8 mutant ovule, showing miss-positioned synergids. The white arrows indicate the egg cell marker expression. Scale bar, 20 μM.
Fig 4.
Bar diagram indicating percentage of aberrant embryo sacs in various mutant backgrounds.
See S1 Table for details. 2n-4n indicates the percentage of gametophytes arrested with 2, 3 or 4 nuclei in an embryo sac that corresponds to a FG6 ovule. “Polar nuclei apart” refers to the percentage of gametophytes with polar nuclei arrested at either end of the central vacuole in a FG7 embryo sac. Extra egg cells and abnormal expression of syn marker were studied in FG7 embryo sacs.
Fig 5.
Expression pattern of the auxin influx carriers AUX1 and LAX1 in the developing embryo sac.
A-C, Detection of AUX1::YFP. D-E, Expression of LAX1 by using the pLAX1:GUS construct (D) or ProLAX1:LAX1-VENUS (E). A,YFP fluorescence is detected at stage FG4 at the micropylar side of the embryo sac. B, After cellularization, at stage FG6, AUX1 is located to egg cell and synergid cell membranes. C, Segregation of YFP signal in a line hemizygous for pAUX1:AUX1:YFP. From the four ovules shown, only two present YFP detectable inside the embryo sac (arrows). The arrowhead indicates an ovule in the same pistile without detectable YFP inside the embryo sac. D, GUS detection driven by the LAX1 promoter shows expression at FG1 stage in the functional megaspore and in the nucellus. E, Expression of LAX1-VENUS in the sporophytic tissues of the nucellus, surrounding the embryo sac micropylar pole at FG2. Scale bar, 20μM.
Table 1.
Quantification of aberrant embryo sacs detected in auxin influx mutants.
Fig 6.
YUC1 overexpressing embryo sacs show extra egg cells and synergid cells at abnormal positions.
A, Confocal image showing DR5::GFP activity localized inside the embryo sac of YUC1 overexpressing female gametophytes. B, GFP signal in A is overlapped with a DIC image. C, A WT embryo sac showing the expression of a specific synergid marker. D, a YUC1 overexpressing embryo sac showing specification of two extra synergid-like cells at a chalazal position (arrows). E, WT embryo sac showing the expression of an egg cell-specific marker. F, A YUC1 overexpressing embryo sac showing expression of the marker in two extra cells at a central position (arrows). Scale bar 20 μM.
Table 2.
Expression of cell specific markers in embryo sacs from pES1::LhG4/+; Op::YUC1/+; GUS/+ plants.