ANI1: A Sex Pheromone–Induced Gene in Ceratopteris Gametophytes and Its Possible Role in Sex Determination

Abstract

Antheridiogen (A CE ) is a pheromone that is required for the development of male gametophytes in the homosporous fern Ceratopteris richardii . Subtractive hybridization of cDNAs isolated from A CE -treated and non-A CE -treated gametophytes was used to isolate genes that are induced by the pheromone. The expression of one gene, ANI1 (for antheridiogen induced), was induced within 3 hr of A CE treatment, but its expression was transient. Patterns of ANI1 expression in wild-type and mutant gametophytes show that ANI1 expression inversely correlates with the predicted activity of one of the sex-determining genes, TRANSFORMER5 ( TRA5 ). These data suggest that ANI1 transcription or transcript accumulation is directly or indirectly prevented by TRA5 in the absence of A CE and that A CE inactivates the TRA5 gene or its product, leading to the upregulation of ANI1 . Cycloheximide (no A CE ) induced the expression of ANI1 , also indicating that ANI1 expression is subject to negative regulation in the absence of A CE . The sequence and inferred protein structure of ANI1 suggest that it is a novel, extracellular protein. The secreted portion of the ANI1 protein potentially forms a ␤ barrel with superficial similarities to lipocalins, which bind small hydrophobic molecules such as pheromones, steroids, and odorants. ANI1 may be an extracellular carrier of A CE that is required to initiate the male program of development as the sexual fate of the young gametophyte is determined. INTRODUCTIONThe haploid gametophytes of many homosporous ferns, including Ceratopteris richardii , develop as either males or hermaphrodites (Figures 1A and 1B). The sex of the gametophyte is determined by the pheromone antheridiogen, or A CE in Ceratopteris (for antheridiogen Ceratopteris) (Schedlbauer and Klekowski, 1972;Banks et al., 1993). Ceratopteris spores develop as male gametophytes with numerous sperm-forming antheridia if they are continuously exposed to exogenous A CE from the time of spore germination. If A CE is removed from the medium surrounding an older male gametophyte, undifferentiated cells of the male prothallus will divide and differentiate a hermaphroditic prothallus, indicating that A CE is required to both initiate and maintain the male program of expression. In the absence of A CE , single spores develop as hermaphroditic gametophytes. These begin to produce and secrete A CE into the surrounding medium after they lose the ability to respond to the pheromone. The loss of sensitivity to A CE coincides with the development of a lateral, multicellular meristem, which is unique to the hermaphrodite ( Figure 1A) (Banks et al., 1993). Exogenous antheridiogen thus performs several functions in the young, sexually undetermined gametophyte: it promotes the differentiation of sperm-forming antheridia and simultaneously suppresses the development of the meristem, the egg-forming archegonia, and the synthesis of antheridiogen (Banks, 1997a).Although the structure of A CE is unknown, several lines of evidence suggest that A CE is a gibb...

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