Summary. Agarose embedding of sunflower (Helianthus annuus L.)hypocotyl protoplasts induces an asymmetric division pattern and subsequent polarized development leading to embryoid formation. We cultured protoplasts in media with different mannitol concentrations. Induction of plasmolysis of agarose-embedded protoplasts by increasing the mannitol concentration lowered the proportion of embryoids formed. This indicates that adhesion sites between the plasma membrane and the agarose matrix are involved in embryoid formation. The involvement of such adhesion sites was tested by incubating embedded protoplasts with RGD peptide. 1 gM RGD beptapeptide reduced embryoid formation by 50% as compared to the control DGR peptide. We also showed that RGD heptapeptide acts on the cytoskeleton by disrupting cortical microtubules. The results are discussed in terms of a model in which the anchorage of the protoplast membrane to the agarose matrix is mediated by RGDbinding proteins connected with microtubules, determining asymmetric division of the cell and polarized development.

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Flowering plants express several LIM-domain proteins related to the animal cystein-rich proteins. The expression of sunflower LIM genes was followed by RT-PCR in cultured sunflower protoplasts. A transcript was detected only for HaWLIM1, but not for the other two genes HaPLIM1 and HaPLIM2. Polyclonal antibodies raised against either full length recombinant HaWLIM1 protein or peptides recognized a 27 kDa polypeptide on Western blots. Immunocytolocalization studies showed that HaWLIM1 is located in the cytoplasm and in the nucleus. In the cytoplasm, HaWLIM1 is localized in punctate structures, distributed along microtubule bundles. Depolymerizing microtubules with oryzalin resulted in a strong modification of the HaWLIM1 cortical pattern. In contrast, treatment of protoplasts with latrunculin B, which disrupts actin filaments, had no effect on HaWLIM1 localization. HaWLIM1 was also located within the nucleus of interphase protoplasts. During mitosis, nuclear labelling was observed in prophase, which decreased in metaphase, disappeared in anaphase, and recovered in telophase. These results suggest a dual role for HaWLIM1: in the cytoplasm, as a component of molecular complexes which may interact with microtubules, and in the nucleus, as a partner of transcription factors during interphase.

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.