No abstract

A. and Alibert, G. 1997. Agarose embedding affects cell wall regeneration and microtubule organization in sunflower hypocotyl protoplasts -Physiol. Plant. 99: 129-134.Sunflower hypocotyl protoplasts (Helianthus annuus L. cv. Emil) divide symmetrically to fonn loosely associated microcolonies when cultured in liquid medium, whereas when embedded in agarose beads they divide asymmetrically to give rise to embryo-like structures. To understand the relationship between protoplast embedding and cell division patterns, we studied the deposition of /i-linked glucan and the dynamics of microtubules during early phases ot culture. After one day in culture, under both culture conditions, a small proportion of the protoplasts had already begun to rebuild a /^glucan cell wall and the process reached completion in all protoplasts after 10 days. Callose deposition was faster in agarose than in liquid medium but it concerned only 30-40% of the protoplasts and was not related to either division type. No marked differences were observed in cortical anays of microtubules. However, in embedded protoplasts perinuclear microtubules formed a well-defined basket around the nucleus; these microtubules were never observed in liquid-cultured protoplasts. A narrow preprophase band was present only in dividing protoplasts cultured in liquid medium. The results suggest that asymmetric division could be related to the lack of a narrow preprophase band and that protoplast embedding enhances nucleation or stabilization of microtubules.

Sunflower hypocotyl protoplasts (Helianthus annuus L. cv. Emil) divide symmetrically to form loosely associated microcolonies when cultured in liquid medium, whereas when embedded in agarose beads they divide asymmetrically to give rise to embryo‐like structures. To understand the relationship between protoplast embedding and cell division patterns, we studied the deposition of β‐linked glucan and the dynamics of microtubules during early phases of culture. After one day in culture, under both culture conditions, a small proportion of the protoplasts had already begun to rebuild a β‐glucan cell wall and the process reached completion in all protoplasts after 10 days. Callose deposition was faster in agarose than in liquid medium but it concerned only 30–40% of the protoplasts and was not related to either division type. No marked differences were observed in cortical arrays of microtubules. However, in embedded protoplasts perinuclear microtubules formed a well‐defined basket around the nucleus; these microtubules were never observed in liquid‐cultured protoplasts. A narrow preprophase band was present only in dividing protoplasts cultured in liquid medium. The results suggest that asymmetric division could be related to the lack of a narrow preprophase band and that protoplast embedding enhances nucleation or stabilization of microtubules.

The cytoskeleton is involved in major developmental events in plant cell growth and differentiation. Nucleation events play a key role in the dynamic and organization of the microtubule (Mt) cytoskeleton. Among many proteins involved in Mt nucleation, γ‐tubulin has been identified as an essential component of the Mt organizing centers (MTOC). In protoplasts, somatic embryogenesis induction has been correlated with remodeling of Mt cytoskeleton. We have investigated the specific developmental expression of γ‐tubulin in Helianthus annuus. Two γ‐tubulin isoforms have been detected by immunoblotting, with bands at 52 and 58 kDa. The larger γ‐tubulin (58 kDa) is present in all the sunflower tissues tested and is associated with the nucleus. The smaller γ‐tubulin (52 kDa), differing from the former at the carboxy‐terminal end, is only present in meristematic and dedifferentiated cells and is not bound to the nucleus. This first demonstration of the presence of two γ‐tubulins in plant cells is discussed in terms of distinct roles in the nucleation and organization of Mts.