Sex determination in Ceratopteris gametophytes

Ferns are extremely useful for studying gametophyte development in plants because their gametophytes develop independently of the sporophyte generation.  In ferns, each tiny gametophyte develops from a single celled spore (the striped spore coat can be seen in the left SEM images below).  In many ferns, the gametophytes are either male or hermaphroditic and are easy to tell apart.  The male makes many sperm-producing antheridia while the hermaphrodite also makes egg-producing archegonia.

The sex of each gametophyte is determined by a pheromone called antheridiogen, or ACE.  This pheromone is made by the hermaphrodite and causes young gametophytes to be male.  This pheromone is likely to be a unique gibberellin.  ABA blocks the Ace response.

To understand how ACE determines the sex these gametophytes, we have identified >100 mutants of Ceratopteris that affect the sex of the gametophyte.  Some of these mutants are shown below.

By tests of epistasis (i.e., comparing the phenotypes of single and different combinations of double or triple mutants), we have ordered these genes into a sex-determining pathway, shown below.  We are now attempting to clone these genes to understand this pathway at the molecular level.

We have also developed a very simple method for knocking-down gene expression in fern gametophytes.  To illustrate, young gametophytes (panel A below) were bombarded with a plasmid into which the Ceratopteris magnesium chelatase gene had been cloned (this gene is required for chlorophyll biosynthesis).  Non-bombarded gametophytes are green (panel B) while bombarded gametophytes are turning white (panel C).  This tool will be useful for discovering the functions of many gametophytically-expressed genes.