Gamete fusion is required to block multiple pollen tubes from entering an Arabidopsis ovule

Abstract

In double fertilization, a reproductive system unique to flowering plants, two immotile sperm are delivered to an ovule by a pollen tube. One sperm fuses with the egg to generate a zygote, the other with the central cell to produce endosperm. A mechanism preventing multiple pollen tubes from entering an ovule would ensure that only two sperm are delivered to female gametes. We use live-cell imaging and a novel mixed-pollination assay that can detect multiple pollen tubes and multiple sets of sperm within a single ovule to show that Arabidopsis efficiently prevents multiple pollen tubes from entering an ovule. However, when gamete-fusion defective hap2(gcs1) or duo1 sperm are delivered to ovules, as many as three additional pollen tubes are attracted. When gamete fusion fails, one of two pollen tube-attracting synergid cells persists, enabling the ovule to attract more pollen tubes for successful fertilization. This mechanism prevents the delivery of more than one pair of sperm to an ovule, provides a means of salvaging fertilization in ovules that have received defective sperm, and ensures maximum reproductive success by distributing pollen tubes to all ovules.

Figure 1

Polytubey is rare in wild type, but increases significantly when gamete fusion fails. (A) hap2(gcs1) enhances polytubey. Differentially marked pollen tubes can be tracked as they grow into an ovule at the micropylar end (M). Representative images of ovules targeted by a DsRed+ pollen tube (left), a GFP+ pollen tube (center), or by two different pollen tubes (right). The number of each type of targeting event observed is also shown (bottom). WT, LAT52:GFP pollen mixed with LAT52:DsRed (both types of pollen are wild type) or hap2-1 (hap2-1/HAP2(GCS1), LAT52:GFP mixed with wild-type, LAT52:DsRed pollen). Scale bars, 20μm. (B) duo1 enhances polytubey. duo1-3/+ heterozygous plants were manually self-pollinated and pollen tubes were analyzed 24 hours later using aniline blue staining[14]. Ovules are shown that have attracted a single pollen tube (left panel), two pollen tubes (center panel), or three pollen tubes (right panel). The percentage of ovules with two or more pollen tubes is plotted for No-0 (wild type, 9 pistils, 260 targeted ovules) and duo1-3/+ (8 pistils, 275 ovules targeted). Error bars represent standard deviation (each pistil is one trial). Rates of polytubey are higher in the No-0 accession (4%) than in ms-1 (Landsberg accession, 1%, Figure 1A) and are significantly enhanced by duo1-3 (36%). (C) Multiple hap2-1 sperm are released into a single ovule. The first panel shows an ovule targeted by a wild-type HTR10:HTR10:mRFP, LAT52:GFP pollen tube. Green signal is released from the burst pollen tube; sperm nuclei (SN, red signal) have fused (F) with the egg cell and central cell nuclei. The second panel shows an ovule targeted by a hap2-1, HTR10:HTR10:mRFP, LAT52:GFP pollen tube. The pollen tube has burst (green signal), but sperm cell nuclei remain unfused (UF). The third panel shows an ovule containing four unfused hap2-1, HTR10:HTR10:mRFP sperm. The last panel shows an ovule containing eight unfused hap2-1, HTR10:HTR10:mRFP sperm. Five sperm nuclei are clearly visible, the others are outside the plane of this image. In this ovule, pollen tube cytoplasm (GFP) appears to fill the area occupied by both synergids. Scale bars, 10μm.

Figure 2

The remaining synergid cell persists longer in ovules targeted by nonfunctional sperm. (A) An unfertilized ovule expressing ACT11:MSI1:GFP. DIC and confocal images are overlaid to highlight the position of the micropyle (M, same in all panels) and GFP accumulation in the egg (EC), central (CC) and synergid (SYN) nuclei. (B–H) Pistils were pollinated with hap2-1/HAP2(GCS1), HTR10:HTR10:mRFP. Representative confocal micrographs are shown of an unfertilized ovule (B), ovules that have received sperm and have a persistent synergid (SYN) (C,E,G), or ovules that have received sperm and both synergids have degenerated (DSYN) (D,F,H). (D) ACT11:MSI1:GFP signal was not detectable in synergids because this image was obtained later in development (note dividing primary endosperm) and significantly after synergid degeneration. (I) Ovules were scored for the number of synergids that remain intact (2, white bar; 1 gray bar; 0 black bar) and whether they had received sperm whose nuclei fused with female nuclei (fused, F) or sperm that remained unfused (U). Data are reported as the percentage of the total number of ovules analyzed (n) at each time point following pollination (hours, hr). Scale bar, 10μm.