A Semi-In Vivo System Reveals Impairment of Cell-Cycle Machinery upon Derailment of Polarity and Cellular Differentiation in In Vitro-Fertilised Egg Cells of Wheat (Triticum aestivum L.)

Cyclin-dependent kinases (CDKs) are central regulators of cell-cycle progression and play essential roles in coordinating DNA replication, mitosis and cytokinesis. Although their involvement in cell proliferation has been extensively investigated, considerably less is known about their contribution to developmental pattern formation during the earliest stages of plant embryogenesis. This question is particularly relevant in angiosperms, where the first zygotic division is intrinsically linked to cell differentiation and formation of the apical–basal body axis. In the present study, the localisation and functional significance of the wheat CDC2 (Cell Division Cycle 2) homologue, p34cdc2, the key regulator of the G2/M transition, were investigated in in vivo- and in vitro-fertilised egg cells of wheat (Triticum aestivum L.). An affinity-purified antibody raised against a synthetic peptide corresponding to the C-terminal region of wheat CDC2 enabled the immunolocalisation of p34cdc2 during zygote development and early embryogenesis. Double-labelling experiments with tubulin showed stage-associated differences in p34cdc2 localisation and its apparent association with the preprophase band, a cytoskeletal structure involved in division-plane determination. Functional analyses showed that pharmacological and antibody-based perturbations were associated with marked changes in early embryonic development. Microinjection of anti-CDC2 and anti-PSTAIRE antibodies into in vitro-fertilised egg cells revealed a critical developmental window shortly after fertilisation during which antibody-mediated interference markedly reduced subsequent cell division. Likewise, inhibition of CDKs by purine derivatives, including olomoucine, disrupted normal morphogenesis and prevented establishment of characteristic embryonic organisation. Oryzalin-mediated disruption of microtubules was accompanied by loss of detectable p34cdc2 localisation to the future division plane, whereas genistein treatment induced severe polarity defects and aberrant embryonic pattern formation. An elaborate semi-in vivo re-implantation system indicated that maternal ovular tissues can support the maintenance of polarity and embryogenic competence during zygote and proembryo development. Taken together, our findings indicate that CDC2-associated processes are closely linked to cell-cycle progression and may be coordinated with polarity establishment and embryonic pattern formation. These results support a model in which interference with CDC2-associated processes can be accompanied by disrupted cell-cycle progression and abnormal embryonic patterning during early wheat embryogenesis.