In vivo analysis of HAP2(GCS1) variants has defined two regions that play distinct roles during HAP2(GSC1)-mediated double fertilization. The protein can be divided into N- and C-terminal regions based on the position of the transmembrane domain. Both regions are essential for function, but different evolutionary constraints are driving their roles in fertilization.
We propose that an extracellular orientation of the N-terminus allows this region to regulate gamete fusion by its interaction with factors on the egg or central cell. This organization is consistent with Hidden Markof Modeling (www.cbs.dtu.dk/services/TMHMM-2.0/
); with the invariant conservation of cysteine residues within the HAP2-GCS1 domain that are predicted to participate in disulfide bonding in an extracellular environment; and with the successful use of N-terminal epitopes to produce antibodies that block Plasmodium reproduction 
. The conserved HAP2-GCS1 domain could, for example, interact with another membrane-bound protein on female gametes facilitating the juxtaposition of the two plasma membranes. Our analysis of interspecific HAP2(GCS1) chimeras is consistent with an extracellular orientation of the N-terminus. Replacement of the Arabidopsis N-terminus with that of a closely related species (Sisymbrium, 89% identical) generated a HAP2(GCS1) variant capable of mediating fusion with Arabidopsis female gametes, but a variant generated with a distantly related sequence failed (rice, 59% identical). These data are consistent with the hypothesis that the egg and central cell express a protein that interacts with HAP2(GCS1) to mediate fusion, and that this protein:protein interaction fails beyond a certain level of sequence divergence in the HAP2(GCS1) N-terminal domain.
Our data also show that HAP2(GCS1) does not contribute to a species level barrier to hybridization. Wind and/or animals indiscriminately pollinate many flowering plant species, so it is important to consider mechanisms that limit hybridization. In some organisms, protein:protein interactions essential for complementary gamete binding and fusion are rapidly co-evolving to enhance reproductive isolation of one species from another 
. We observe that the N-terminus of Sisymbrium, but not rice HAP2(GCS1), can mediate fertilization with Arabidopsis female gametes. Arabidopsis and Sisymbrium are in the Brassica family, but belong to distinct tribes 
. Thus, our data suggest that Arabidopsis female gametes can distinguish between the N-terminal sequences of HAP2(GCS1) from Arabidopsis and distantly related rice, but cannot discriminate Arabidopsis from closely related Sisymbrium. The recent finding that pollen tubes are attracted to ovules by small proteins with species-specific activity 
supports a model that barriers prior to gamete-gamete interaction account for species-level discrimination in flowering plants, potentially leaving the proteins involved in gamete-gamete interactions to evolve without diversifying selection.
Positive charge, not primary amino acid sequence, is the C-terminal characteristic conserved among HAP2(GCS1) orthologs and our data show that positive charge is required for function. Unlike the protein:protein interactions proposed for the N-terminus, the intracellular C-terminus may be functioning through electrostatic interactions with negatively charged molecules (e.g. the inner face of the plasma membrane) that favor membrane fusion. Positively charged domains located on the intracellular domain of fusion-associated small transmembrane (FAST) proteins have been implicated in fusion of host cells by non-enveloped viruses 
Flowering plant C-termini are enriched in histidine whereas other positively charged amino acids (arginine and lysine) are prevalent in other orthologs 
, suggesting that selection for one class of charged amino acids over another has shaped the evolution of HAP2(GCS1) in different eukaryotes. These three positively charged amino acids were functionally interchangeable in our Arabidopsis experiments. In nature, however, differences in the composition of the C-terminal domain may have been selected to meet the unique demands of the reproductive systems that use HAP2(GCS1). Under physiologic pH (e.g. pH 5–7), histidine exists in either a protonated or neutral form (pKa
6.08) whereas lysine (pKa
10.5) and arginine (pKa
12.0) are always protonated. Perhaps the difference in sperm delivery mechanisms between flowering plants and other eukaryotes selected for the bimodal charge state of histidine. Flowering plant sperm develop within the pollen cytoplasm and are delivered to the ovule by a pollen tube. hap2-1
pollen tubes have a reduced ability to target ovules compared to wild type, suggesting that in flowering plants, HAP2(GCS1) may have a role in pollen tube guidance that is distinct from its essential role in gamete fusion 
. Future experiments will test these hypotheses by determining if HAP2(GCS1) variants with modified C-termini can complement the pollen tube guidance defect observed when hap2-1
pollen tubes compete with wild-type pollen tubes for access to ovules. Pollen tubes burst upon arrival at the ovule, exposing sperm to the extracellular environment. It will be interesting to determine if this change in environment results in a drop in sperm pH that activates HAP2(GCS1) function.
hap2-1 sperm expressing a HAP2(GCS1) variant with a neutralized C-terminus (AtN•AtC mut øΔ) had significantly reduced fertility. Double fertilization occurred in only ~7% of the ovules we analyzed, while many ovules remained unfertilized (~40%). A large portion of the ovules contained products of single fertilization events (~23% embryo-only, 8% endosperm-only) that fail to complete seed development. These results highlight a unique advantage of flowering plants for the study of gamete fusion: the outcomes of two distinct fertilization events, both requiring HAP2(GCS1) function, can be observed independently. This situation provides a sensitive means to detect reduced fusion efficiency. We consistently observed fertilization of only one female gamete when two hap2-1 sperm expressing the AtN•AtC mut øΔ HAP2(GCS1) variant were delivered to an ovule, specifically detecting more single fertilizations with the egg (embryo-only) than the central cell (endosperm-only). This suggests that sperm:central cell fusion may require more HAP2(GCS1) activity or that central cell fusion is particularly sensitive to the C-terminal charge of HAP2(GCS1).
All evidence to date indicates that HAP2(GCS1) has an essential role in fertilization 
, but its exact function remains unknown. Observations made in Chlamydomonas suggest it is required for gamete fusion because gamete attraction and binding/juxtaposition of membranes are normal in HAP2(GCS1)
gametes, yet membranes fail to fuse 
. One hypothesis is that HAP2(GCS1) directly catalyzes membrane fusion 
. While HAP2(GCS1) does not share primary sequence with known fusogenic proteins, it shares features with the FAST proteins of non-enveloped viruses. FAST proteins have a single transmembrane domain, a conserved, extracellular N-terminus and a variable C-terminus that is positively charged 
. A cell expressing FAST proteins can fuse with a non-expressing neighboring cell 
, so like HAP2(GCS1), the requirement for FAST proteins in fusion is asymmetric. Thus, by virtue of their common attributes, HAP2(GCS1) and FAST proteins may use a similar mechanism to catalyze membrane fusion.
We have mapped the key domains of HAP2(GCS1) and propose a model in which the N-terminus functions by interacting with female gamete-expressed proteins and the C-terminus is required to interact with the plasma membrane through its positive charge. By analogy to known fusogenic proteins, we propose that these interactions bring gamete membranes into close proximity, destabilize the phospholipid bilayer, and generate membrane structures favoring their fusion 
. Future studies designed to directly assess the ability of HAP2(GCS1) to catalyze membrane fusion will be required to test this model and to elucidate the biochemical function of this ancient reproductive protein.