Establishment of the Growth Axis and Alignment of the Division Plane in Pelvetia Zygotes

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Establishment of embryonic axes is fundamental to the development of eukaryotic organisms, yet very little is known about this process in plants because the embryo is retained inside maternal tissues and is difficult to isolate. In contrast, zygotes of the brown algae, Fucus sp. and Pelvetia sp., are easily harvested from seawater and have long served as model organisms for investigation of polarity establishment. For the past nine years the Kropf lab has been studying the cell biology and physiology of polarization in P. compressa zygotes focusing primarily on the roles of cytosolic ions and the cytoskeleton. Based upon this research, and that from other laboratories, the Kropf lab has formulated a working model to account for establishment of the growth axis and the precise orientation of the division plane. Briefly, this model postulates that the rhizoid pole of the growth (rhizoid-thallus) axis is marked by a patch of cortical F-actin and that the division plane is determined by the orientation of centrosomes on the nuclear envelope. Sperm entry is postulated to induce both axes by causing assembly of an F-actin patch at the sperm entry site and by introducing centrioles into the egg cytoplasm. Over the first few hours of development, the F-actin patch, and hence the growth axis, can be repositioned in response to environmental cues. The centrosomal axis does not respond to environmental cues and therefore the two axes develop independently of one another. The PI speculates that the F-actin patch at the rhizoid pole defines a target site for secretion, and that polar secretion of molecules locally into the rhizoid plasma membrane and cell wall amplifies the nascent growth axis. After growth begins at the rhizoid pole, and just prior to first mitosis, the centrosomal axis rotates into parallel alignment with the growth axis. The spindle poles form from the aligned centrosomes and cytokinesis bisects the spindle, ensuring that the division plane is transverse to the growth axis.The proposed research will test the salient aspects of this working model. The primary objectives are to:1) determine whether sperm entry induces an initial polarity2) demonstrate that a patch of F-actin marks the rhizoid pole of the axis3) investigate the mechanism of polar secretion4) analyze the role of microtubules (MTs) in rotational alignment of the centrosomes5) clarify the role of plasma membrane-cell wall adhesions in rotational alignment6) investigate regulation of the cytoskeletal arrays by cytosolic H+ activityConfirmation of our working model will provide the first comprehensive picture of the role of the cytoskeleton in polarity establishment and expression in fucoid zygotes, and will initiate important studies on polar secretion and cytoskeletal regulation by H+. This study will also provide a paradigm for future investigations of polarity establishment in higher plant embryos.