Agarose embedding affects cell wall regeneration and microtubule organization in sunflower hypocotyl protoplasts

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.

Auxin deprivation induces a developmental switch in maize somatic embryogenesis involving redistribution of microtubules and actin filaments from endoplasmic to cortical cytoskeletal arrays

“…For instance, sunflower protoplasts cultured in liquid medium form loosely associated cells and do not embark on the embryogenic pathway. However, when the same cells are embedded in agarose, they start to deposit callose at their reforming cell walls, assemble numerous perinuclear microtubules, and enter the embryogenic pathway (Caumont et al 1997). It would be interesting to know whether these callosic cell walls also accumulate some AGPs.…”

“…They are also abundant in Arabidopsis mutants with affected cell growth polarity and morphogenesis (Hauser et al 1995;Traas et al 1995). Numerous endoplasmic microtubules are present in cells treated with auxin (for maize roots see BaluÐka et al 1996b) or subjected to diverse stresses including cold (BaluÐka et al 1993), heat (Hause et al 1993;Gervais et al 2000), mechanical stress (Caumont et al 1997), and aluminium (Sivaguru et al 1999). Similarly, during sporogenesis (Dickinson and Sheldon 1984), endosperm development (Brown and Lemmon 1988;Brown et al 1994), and Rhizobium infection (Timmers et al 1999), centered nuclei organize almost all microtubules in the form of radial arrays.…”

“…In all these situations, cortical microtubules are usually depleted and/or disassembled. However, little is known about cytoskeletal organization of embryogenically competent cells during embryogenic induction and progress (Caumont et al 1997). …”

RGD-mediated membrane-matrix adhesion triggers agarose-induced embryoid formation in sunflower protoplasts

“…Thus, physical constraints cannot solely explain the protoplast division pattern observed in agarose cultures. Recently, in a study of the cytoskeleton of agaroseembedded protoplasts, Caumont et al (1997) reported that the agarose matrix interacts with cortical microtubules and stabilizes the cytoskeleton. In animal cells, it is well known that several plasma membrane proteins are involved in linkages of the cytoskeleton to the extracellular matrix, the most prominent and best studied matrix receptors belonging to the integrin family (Hynes 1992 (Henry et al 1996).…”

A bacterial artificial chromosome (BAC) library for sunflower, and identification of clones containing genes for putative transmembrane receptors

“…In sunflower, somatic embryogenesis is also an important tool, enabling the analysis of embryo development outside the maternal environment (Scheres and Benfey 1999;Vroemen et al 1999). An experimental system in which, depending on the culture conditions, an isolated cell (protoplast) can give rise to somatic tissues or to embryo-like structures has also been developed in sunflower (Caumont et al 1997).…”