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Originally published in Science Express on 4 June 2009
Science 26 June 2009:
Vol. 324. no. 5935, pp. 1684 - 1689
DOI: 10.1126/science.1167324
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Research Articles

Auxin-Dependent Patterning and Gamete Specification in the Arabidopsis Female Gametophyte

Gabriela C. Pagnussat1,*, Monica Alandete-Saez1, John L. Bowman1,2 and Venkatesan Sundaresan1,3,{dagger}

1 Department of Plant Biology, University of California, Davis, CA 95616, USA.
2 School of Biological Sciences, Monash University, Melbourne, Victoria 3800, Australia.
3 Department of Plant Sciences, University of California, Davis, CA 95616, USA.


Figure 1
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  		Fig. 1. Expression of the synthetic reporters DR5::GFP during female gametophyte development. (A) Scheme showing the developmental stages during wild-type female gametophyte development in Arabidopsis. Ant indicates antipodal cells; Cc, central cell; Ec, egg cell; Fg, female gametophyte; Fm, functional megaspore; ii, inner integument; nu, nucellus; oi, outer integument; Pn, polar nuclei; and Syn, synergid. (B), At FG1, the signal is strongly detected in the nucellus, outside the embryo sac. Scale bar indicates 25 µm, and the developing embryo sac is delimited by a dashed line. (C) At FG3 stage, the signal is detectable inside the embryo sac at the micropylar pole. (D) As the female gametophyte continues to develop, a strong DR5::GFP signal is localized to the micropylar end of the embryo sac by stage FG4. Small black arrowheads indicate nuclei inside the developing embryo sac. (E) A DR5::GFP activity maximum at the micropylar end of the embryo sac could be detected up to FG5 stage. (F) At FG6 stage the distribution of the DR5::GFP signal becomes less polarized. Signals outside the developing embryo sac in (C), (E), and (F) correspond to vascular tissues in the sporophytic ovule, particularly the funiculus.

 

Figure 2
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  		Fig. 2. When several ARF genes were down-regulated with an artificial miRNA expressed specifically in the embryo sac identity, defects in micropylar cells were observed. Scale bars, 25 µm. (A) DIC image of a wild-type (WT) embryo sac. Ccn, central cell nucleus; Ec, egg cell; and Syn, synergid. (B) Scheme of a WT female gametophyte. (C) Mature embryo sac showing an abnormal egg cell (Ab. Ec) with a centrally located nucleus. (D) Embryo sac showing unfused polar nuclei (Pn) and an abnormal synergid (Ab. Syn) cell, with a nucleus located toward the chalazal end of the embryo sac. (E) Embryo sac exhibiting two egglike cells. EcL, egg cell–like cell. (F) Embryo sac showing three cells morphologically similar to egg cells. (G) Expression of an egg cell–specific marker in a WT embryo sac. The developing embryo sac (Es) is delineated by a dashed line. (H) Expression of an egg cell–specific marker in all three micropylar cells of an ARF–down-regulated embryo sac. Mc, micropylar cell.

 

Figure 3
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  		Fig. 3. Expression of YUCCA genes in transgenic plants carrying YUC1::GUS and YUC2::GUS constructs suggests a role for auxin biosynthesis in gametophyte development. (A to F) Expression of YUC1 (A to C) and YUC2 (D to F) in wild-type embryo sacs. Scale bars, 25 µm. (A) YUC1 expression in the nucellus at FG1 stage. (B) YUC1 expression at the micropylar end of the embryo sac at stage FG3. (C) YUC1 expression at the micropylar end of the embryo sac at stage FG4. (D) YUC2 expression in the nucellus at FG1 stage. (E) YUC2 expression at the micropylar end of the embryo sac at stage FG3. (F) YUC2 expression at the micropylar end of the embryo sac at stage FG4.

 

Figure 4
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  		Fig. 4. Phenotype of YUC1-overexpressing embryo sacs and two alternative models for auxin-dependent cell specification in the female gametophyte. Scale bars, 25 µm. (A) DR5::GFP activity is localized in the whole embryo sac (Es) of YUC1-overexpressing embryo sacs at FG5. (B) Polarized DR5::GFP activity detected in a WT embryo sac at FG5. Ccn, central cell nucleus; Syn, synergid cells; and Syn LC, synergid-like cell. (C) A YUC1-overexpressing embryo sac with a synergid-like cell at a position in which the egg cell is usually located. (D) WT embryo sac showing the expression of a specific synergid marker. Ch, chalazal end of the embryo sac, and mi, micropylar end of the embryo sac. (E) Expression of the synergid marker in a YUC1-overexpressing embryo sac shows signal in the positions of both the synergid cells and the antipodal cells. (F) Expression of the synergid marker is detected in cells at the positions of synergids, egg cell, and central cell in a YUC1-overexpressing embryo sac. (G) Wild-type embryo sac showing the expression of an egg cell (E.C)–specific marker. (H) YUC1-overexpressing embryo sac exhibiting expression of the egg cell marker in a cell at the position of the egg cell and a cell at the antipodal position. (I) YUC1-overexpressing embryo sac showing expression of the egg cell marker only in a cell at the antipodal position. (J) Mature and unfertilized YUC1-overexpressing embryo sac from an emasculated flower showing surviving antipodal cells (S Ant) 2 days after emasculation. (K) Two pollen tubes (Pt) enter the micropyle of a YUC1-overexpressing embryo sac and continue to grow within the embryo sac. F, funiculus. (L) Fertilized YUC1-overexpressing embryo sac showing no endosperm development and a structure that morphologically resembles a zygote at the position of the central cell. EcL, egg cell–like cell; Z, zygote, and ZL, zygotelike cell.

 

Figure 5
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  		Fig. 5. Models for patterning of the female gametophyte. (A to D) Auxin gradient model for patterning. (A) At FG1 stage, the auxin source is sporophytic and is derived from the nucellus. (B) Subsequently, a secondary auxin source is specified locally within the gametophyte at the micropylar pole. (C) Auxin gradient with a maximum at the micropylar pole of the embryo sac. (D) Auxin concentration determines cell fates, with the highest auxin concentrations specifying synergids (Syn), followed by egg cells (E.C), and the lowest auxin resulting in antipodal (Ant.) cells. Central C., central cell. (E to H) Model for patterning by a nonauxin molecule acting through auxin. (E) Nucellus generates a signal (block arrow), which might originate from the auxin source. (F) This signal induces a factor X at the micropylar pole, which then induces auxin synthesis by activating YUCCA gene expression in the nucleus located at that pole. (G) Continued synthesis of auxin at micropylar pole. (H) Two of the micropylar nuclei are in the high auxin zone and differentiate into synergids, whereas the third micropylar nucleus in lower auxin differentiates into the egg cell. In this model, the central cell and antipodal cell fates could be specified by auxin-dependent or auxin-independent mechanisms.

 

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