Identification of three proteins involved in fertilization and parthenogenetic development of a brown alga, Scytosiphon lomentaria.

Abstract

Metabolic pathways of cell organelles may influence the expression of nuclear genes involved in fertilization and subsequent zygote development through a retrograde regulation. In Scytosiphon lomentaria, inheritance of chloroplast is biparental but mitochondria are maternally inherited. Male and female gametes underwent different parthenogenetic outcomes. Most (>99%) male gametes did not differentiate rhizoid cells or survived beyond four-cell stage, while over 95% of female gametes grew into mature asexual plants. Proteomic analysis showed that the protein contents of male and female gametes differed by approximately 1.7%, 12 sex-specific proteins out of 700 detected proteins. Three sex-specific proteins were isolated and identified using CAF-MALDI mass spectrometry and RACE-PCR. Among them, a male gamete-specific homoaconitate hydratase (HACN) and a female gamete-specific succinate semialdehyde dehydrogenase (SSADH) were predicted to be the genes involved in mitochondrial metabolic pathways. The expression level of both mitochondrial genes was dramatically changed at the fertilization event. During parthenogenetic development the male-specific HACN and GTP-binding protein were gradually down-regulated but SSADH stayed up-regulated up to 48h. To observe the effect of chemicals on the expression of these genes, male and female gametes were treated with γ-aminobutyric acid (GABA), hydrogen peroxide and L-ascorbic acid. Among them GABA treatment significantly reduced SSADH gene expression in female gamete but the same treatment induced high upregulation of the gene in male gamete. GABA treatment affected the behavior of gametes and their parthenogenetic development. Both gametes showed prolonged motile stage, retarded settlement and subsequent parthenogenetic development. Our results suggest that male and female gametes regulate mitochondrial metabolic pathways differentially during fertilization, which may be the reason for their physiological and behavioral differences.