Arabidopsis HAP2(GCS1) is a sperm-specific gene required for pollen tube guidance and fertilization

Abstract

In flowering plants, sperm cells develop in the pollen cytoplasm and are transported through floral tissues to an ovule by a pollen tube, a highly polarized cellular extension. After targeting an ovule, the pollen tube bursts, releasing two sperm that fertilize an egg and a central cell. Here, we identified the gene encoding Arabidopsis HAP2, demonstrating that it is allelic to GCS1. HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to ovules. We identified an insertion (hap2-1) that disrupts the C-terminal portion of the protein and tags mutant pollen grains with the ␤-glucuronidase reporter. By monitoring reporter expression, we showed that hap2-1 does not diminish pollen tube length in vitro or in the pistil, but it reduces ovule targeting by twofold. In addition, we show that the hap2 sperm that are delivered to ovules fail to initiate fertilization. HAP2 is predicted to encode a protein with an N-terminal secretion signal, a single transmembrane domain and a C-terminal histidine-rich domain. These results point to a dual role for HAP2, functioning in both pollen tube guidance and in fertilization. Moreover, our findings suggest that sperm, long considered to be passive cargo, are involved in directing the pollen tube to its target.

An EAR-Dependent Regulatory Module Promotes Male Germ Cell Division and Sperm Fertility in Arabidopsis

“…In six observations of single gamete discharge, fertilization of the egg or central cell did not occur (Figures 6H to 6M; Supplemental Movie 1 and 2), similar to previous observations of mutant duo3 germ cells and gcs1/hap2 sperm cells (Kasahara et al, 2012). Given that daz1 daz2 double mutant gametes are not competent for fertilization, we monitored gamete differentiation by introducing markers for two DUO1 target genes, the male gamete-specific histone H3.3 variant HTR10/MGH3 (Okada et al, 2005) and the sperm plasma membrane protein GCS1/HAP2, which is required for gamete fusion (Mori et al, 2006;von Besser et al, 2006). All daz1 daz2 germ cells expressed ProHTR10:H2B-GFP ( Figure 7A), although quantification of GFP fluorescence indicated a significant reduction in expression compared with sperm cells ( Figure 7B).…”

“…The daz1 and daz2 alleles were obtained from the SALK T-DNA insertion collection (daz1-1, SALK_058012; daz1-2, SALK_151422; daz2-1, SALK_101906) (Scholl et al, 2000). Seeds of the ProHAP2:HAP2-eYFP marker line (von Besser et al, 2006) were kindly provided by Mark Johnson. Transgenic ProCYCB1;1: MDB-GFP, ProHTR10:H2B-GFP, and ProDUO1:CYCB1;1 lines have been described previously (Brownfield et al, 2009a(Brownfield et al, , 2009b.…”

Comparative genomics in Chlamydomonas and Plasmodium identifies an ancient nuclear envelope protein family essential for sexual reproduction in protists, fungi, plants, and vertebrates

“…In spite of the many cellular events common to fertilization and zygote development in almost all eukaryotes, only recently was the first gamete-specific gene identified that is conserved and essential for fertilization in broadly disparate organisms. The gamete membrane protein HAP2(GCS1) is now known to be essential for gamete fusion in the isogamous, unicellular green alga Chlamydomonas; the unicellular malaria organism Plasmodium; and the higher plant Arabidopsis (Mori et al 2006;von Besser et al 2006;Hirai et al 2008;Liu et al 2008). Based on our results from Chlamydomonas and Plasmodium, the current model is that HAP2 is a gamete membrane fusogen in all three organisms and presumably in the many other protists, plants, and multicellular animals that possess it (Mori et al 2006;von Besser et al 2006;Hirai et al 2008;Liu et al 2010;Wong and Johnson 2010).…”

Cis-Regulatory Elements Determine Germline Specificity and Expression Level of an Isopentenyltransferase Gene in Sperm Cells of Arabidopsis

“…For example, DUO1 is a MYB transcription factor that is essential to male germline transcription and identity, with specific expression in generative and sperm cells of Arabidopsis (Rotman et al, 2005;Brownfield et al, 2009a;Borg et al, 2011). The highly conserved GCS1/ HAP2 gene encoding a secreted membrane protein is essential for fertilization in a myriad of other eukaryotes as well (Mori et al, 2006;von Besser et al, 2006;Liu et al, 2008). Additional male gamete-selective genes have been identified and characterized in lily and Arabidopsis (Engel et al, 2005;Okada et al, 2005aOkada et al, , 2005bBrownfield et al, 2009aBrownfield et al, , 2009b.…”

“…The expression of LGC1 is exclusively restricted in male gamete cells (Xu et al, 1999). Another lily gene, GCS1, and its Arabidopsis homolog HAP2 are expressed in sperm cells and are essential for fertilization (Mori et al, 2006;von Besser et al, 2006). Sperm cell-expressed genes GAMETE EXPRESSED 1 (GEX1) and GEX2 were identified in maize sperm cell-specific transcripts, and homologous Arabidopsis genes were designated AtGEX1 and AtGEX2 (Engel et al, 2005).…”