<i>Arabidopsis</i> gamete fusion requires presentation of hydrophobic residues within the amphipathic HAP2 αF helix.

(A) Schematic of male gamete delivery and double gamete fusion within an Arabidopsis flower. Four anthers are shown surrounding the pistil, which contains two columns of ovules; petals and sepals are not depicted. Two pollen tubes (black lines) are shown targeting ovules. The inset illustrates a pollen tube approaching a female gametophyte, which develops within the ovule. A pair of sperm cells and the pollen tube nucleus are shown within the pollen tube; synergid cells (“SYN”), egg cell (“EC”), central cell (“CC”), and three antipodal cells (dark blue) are shown within the female gametophyte. (B) A schematic showing a pollen tube that has burst into a synergid, propelling the sperm to the site of fusion with the egg or central cell. (C) Arrows highlight the fusion of one sperm with the egg and the other with the central cell. (D) Assay for HAP2 function in vivo using transmission of hap2-2 (T-DNA insertion carries kanamycin resistance, “KanR”). hap2-2/+ plants carrying WT or variant HAP2:YFP transgenes were produced. These plants will generate four pollen/sperm genotypes. Endogenous HAP2 (black) or introduced HAP2:YFP construct (purple/yellow) are shown at the sperm surface. The fertility outcome for each sperm genotype is indicated; a functional copy of HAP2 (either endogenous or a functional HAP2:YFP variant) must be expressed by sperm to produce a viable seed. Only hap2-2 sperm that carry a functional HAP2:YFP variant transgene will sire KanR progeny when outcrossed to ms1 females. Expression of a fully functional HAP2:YFP variant will result in 33% KanR seedlings. (E) The Arabidopsis (“At”) and rice (“Os”) HAP2 membrane interaction surfaces including the αF helix are shown; the region chosen for mutagenesis is identified with a box. (F) Quantitative analysis of effect of HAP2:YFP variants on hap2-2 transmission (see S2 Data). Each data point (circle) represents the percentage of progeny from a single cross-pollination that inherited the hap2-2 allele; the mean is indicated with a red diamond. A minimum of three independent transgenic lines were analyzed for each HAP2:YFP variant (see Materials and methods). HAP2:YFP variants tested are indicated on the x-axis: none, no transgene; others defined in text; p-values from the Wilcoxon rank sum test comparing each variant to AtHAP2 are indicated: *p p HAP2, HAPLESS 2; ms1, male sterile 1; ns, not significant; T-DNA, transfer DNA; WT, wild type; YFP, yellow fluorescent protein