Recovery of microtubules on the blepharoplast of Ceratopteris spermatogenous cells after oryzalin treatment

Abstract

Most land plants have ill-defined microtubule-organizing centers (MTOCs), consisting of sites on the nuclear envelope or even along microtubules (MTs). In contrast, the spermatogenous cells of the pteridophyte Ceratopteris richardii have a well-defined MTOC, the blepharoplast, which organizes MTs through the last two division cycles. This allows a rare opportunity to study the organization and workings of a structurally well-defined plant MTOC. In this study, antheridial plants were treated with levels of oryzalin that cause complete MT loss from the cells containing blepharoplasts. The oryzalin was then washed out and plants were allowed to recover for varying amounts of time. If the spermatogenous cells were fixed prior to washing out, the blepharoplasts had an unusual appearance. In the matrix (pericentriolar) material where MT ends are normally found, clear areas of about the diameter of MTs were seen embedded in a much deeper matrix, made more obvious in stereo pairs. Occasionally, the matrix material was highly distended, although the basal body template cylinder morphology appeared to be unaltered. The blepharoplasts often occurred as clusters of 2 or 4, indicating that blepharoplast reproduction is not affected by the lack of MTs, but that their movement to the poles is. Gamma (γ) tubulin antibodies labeled the edge of the blepharoplast in areas where the pits are located, indicating that these might be sites for MT nucleation. After wash out, the new MTs always re-appeared on the blepharoplast and the recovery occurred within an hour of washout. MT lengths increased with increasing washout time and were indistinguishable from untreated blepharoplasts after 24 h of recovery. After washout, arrays formed in new sperm cells such as the spline and basal bodies were often malformed or present in multiple copies, as were the blepharoplasts in these cells prior to wash out. These data indicate that the blepharoplast serves as the site of MT nucleation and organization even after complete MT de-polymerization.