The peroxin loss-of-function mutation abstinence by mutual consent disrupts male-female gametophyte recognition

Abstract

In eukaryotes, fertilization relies on complex and specialized mechanisms that achieve the precise delivery of the male gamete to the female gamete and their subsequent union [1-4]. In flowering plants, the haploid male gametophyte or pollen tube (PT) [5] carries two nonmotile sperm cells to the female gametophyte (FG) or embryo sac [6] during a long assisted journey through the maternal tissues [7-10]. In Arabidopsis, typically one PT reaches one of the two synergids of the FG (Figure 1A), where it terminates its growth and delivers the sperm cells, a poorly understood process called pollen-tube reception. Here, we report the isolation and characterization of the Arabidopsis mutant abstinence by mutual consent (amc). Interestingly, pollen-tube reception is impaired only when an amc pollen tube reaches an amc female gametophyte, resulting in pollen-tube overgrowth and completely preventing sperm discharge and the development of homozygous mutants. Moreover, we show that AMC is strongly and transiently expressed in both male and female gametophytes during fertilization and that AMC functions in gametophytes as a peroxin essential for protein import into peroxisomes. These findings show that peroxisomes play an unexpected key role in gametophyte recognition and implicate a diffusible signal emanating from either gametophyte that is required for pollen-tube discharge.

Figure 1. Siliques of amc/+ mutant lack homozygous individuals

(A) Schematic drawing of male and female gametophytes during fertilization in Arabidopsis thaliana. The pollen tube (PT) enters the ovule through the micropyle, an opening leading to the seven-celled haploid female gametophyte and reaches the receptive synergid cell in which it will discharge its sperm cells, a process called pollen tube reception. During double fertilization, one sperm cell fuses with the egg cell, which will give rise to the zygote, while the second sperm will fertilize the central cell to induce endosperm formation. Scale Bar is 20 μm. (B) In contrast to wild-type siliques (top), siliques of self-pollinated amc/+ plants frequently contained degenerating ovules (bottom, white arrows). (C) Exon/Intron organization of the APM2/AMC locus (At3g07560) and position of the T-DNA in the amc/+ mutant. Positions of the primers used to genotype the amc/+ mutant and position of the point mutation in the weak apm2 allele [15] are indicated.

Figure 2. The amc mutation disrupts pollen tube growth arrest and discharge

(A–H) Examination by confocal microscopy of YFP-expressing pollen 19–24h after manual pollination during wild-type (A–D) and amc mutant (E–H) pollen/ovule interactions. Fluorescence from the YFP expressed in pollen tubes is shown in green while the autofluorescence from the ovule tissues is shown in magenta. (A) A wild-type pollen tube reaching the micropylar side of the synergid cells. (B,C) Two examples of wild-type pollen tube discharges. At the extremity of the PT, the fluorescence assumes the shape of the receptive synergid cell in which the pollen tube has released its content. (D) Close-up of (C). (E–G) Three examples of continued growth of presumably an amc pollen tube reaching an amc ovule (see Results and Discussion). The mutant pollen tube while reaching the micropylar part of the synergid cells does not discharge. It keeps growing by coiling and branching (F,G) and the pollen tube tips can be spotted past the synergid cells of the mutant ovule (E,F). Among these mutant interactions, two pollen tubes can be frequently observed within a presumed amc ovule (E, white arrows). (H) Close-up image of the coiled pollen tube shown in (G). Note how the pollen tube coils and branches making the fluorescence outlines rougher than in (D). Scales bars are 23 μm for A–C, E–G and 7 μm for D,H.

Figure 3. APM2/AMC expression pattern supports its role during fertilization

(A) Semi-quantitative RT-PCR (bottom, 31 cycles) and quantitative real-time (RT)-PCR (top) analyses of APM2/AMC expression in different organs/tissues. ACTIN7 (At5g09810) was used as a control for RT-PCR analysis. For quantitative real-time (RT)-PCR experiments, Clathrin (At4g24550) was used as an internal control and each data point indicates the average of three independent experiments ± SEM. In line with the microarray data (Figure S4A), these analyses indicated that APM2/AMC is preferentially expressed in mature pollen grains. (B–F) Histochemical localization of GUS reporter gene expression driven by the APM2/AMC promoter in pollen grains (B) and during fertilization (C–F). (B) one hour GUS-stained pAMC-GUS pollen (top) and untransformed wild-type pollen (bottom) grains. Scale bar is 5 μm. (C–F) GUS expression in a mature female gametophyte before (C), during (D), 24h after (E) and 48h (F) after fertilization (12h GUS staining). During fertilization (D), both male (arrow) and female (asterisk) gametophytes are strongly stained. Note how strongly the GUS activity dropped after fertilization (E, F). Scale bars are 28 μm for C–F. See also Supplemental Data and Figure S4F.

Figure 4. PTS1-dependent protein import into peroxisomes is completely impaired in amc mutant pollen

Examination by confocal microscopy of pollen tetrads of wild-type (A) and amc/+ (B) plants heterozygous for the peroxisomal pLAT52-CFP-PTS1 marker. Left panels, bright light; right panels, CFP filter. Scale bar is 10 μm. (A) In wild-type tetrads, all the fluorescent pollen grains (half of the total grains) exhibited a fluorescence with a peroxisome-like punctuated pattern (white arrows) indicative of a normal PTS1-dependent import into peroxisomes. (B) In amc/+ tetrads, in a 1:1 expected ratio for segregation of the fully penetrant amc mutation, fluorescent pollen grains exhibited fluorescence either in a punctuated pattern as in wild-type (arrow) or exclusively in the cytosol (arrowhead). In this latter case, note how peroxisomes can no longer be discerned, indicating complete impairment of PTS1-dependent import into peroxisomes (see also Figure S7 for pollen tubes growing in vitro).