THE ROLE OF SPERMIDINE IN THE REGULATION OF DEVELOPMENT AND DIFFERENTIATION IN SPERMATIDS OF <italic>MARSILEA VESTITA</italic>

Abstract

Spermiogenesis in the microspore of the water fern,<italic> Marsilea vestita</italic>, is a rapid process where a dry microspore containing a single cell undergoes nine successive mitotic divisions to produce 32 spermatids and seven sterile cells. Immediately after the dry microspore is placed into water, cytoplasmic movements precede the first mitotic division; a number of proteins and mRNAs aggregate into zones that later become the spermatogenous initials of the gametophyte. Development is driven by the regulated translation of stored mRNA with little or no new transcription (Hart and Wolniak, 1999). The pattern of translation in the gametophyte is ordered precisely spatially and temporally, which indicates that certain proteins are required in specific locations at specific stages of development. Spermatid differentiation involves the <italic>de novo</italic> synthesis of 140 basal bodies, the remodeling and condensation of nuclear chromatin and then, nuclear elongation. A complex cytoskeletal structure, the multilayered structure (MLS), is formed at the anterior end of the cell and extends the length of the elongated gamete, and apparently functions in cell and nuclear elongation. This document focuses on the role of kinesin motor proteins and spermidine in the regulation of gametophyte development and spermatid differentiation. Blocking the translation of various kinesin isoforms with RNA interference (RNAi) resulted in arrested development at distinct time points. Centrin and tubulin immunolabeling showed different defects in basal body and microtubule ribbon formation, respectively, with the silencing of specific kinesins. The reduction of spermidine levels in the gametophytes by silencing proteins responsible for its synthesis and transport reveals the involvement of the polyamine in gametophyte cell cycle regulation and in spermatid maturation. In addition, drug inhibition of spermidine synthesis later in development highlighted the importance of its involvement in chromatin remodeling and nuclear elongation. Immunolabeling of spermidine and <italic>in situ</italic> hybridization assays for its synthesizing enzyme, spermidine synthase, indicated that spermidine levels are controlled in gametophytes by the regulated translation of spermidine synthase. The regulated levels of spermidine in the gametophyte is key to its function as a developmental regulator; spermidine appears to participate in multiple cellular processes in a concentration dependent manner.