Abstract
Antheridiogen (ACE) is a pheromone that is required for the development of male gametophytes in the homosporous fern Ceratopteris richardii. Subtractive hybridization of cDNAs isolated from ACE-treated and non-ACE-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 ACE 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 ACE and that ACE inactivates the TRA5 gene or its product, leading to the upregulation of ANI1. Cycloheximide (no ACE) induced the expression of ANI1, also indicating that ANI1 expression is subject to negative regulation in the absence of ACE. 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 beta barrel with superficial similarities to lipocalins, which bind small hydrophobic molecules such as pheromones, steroids, and odorants. ANI1 may be an extracellular carrier of ACE that is required to initiate the male program of development as the sexual fate of the young gametophyte is determined.