Henri Barthou

RGD-MEDIATED MEMBRANE-MATRIX ADHESION TRIGGERS AGAROSE-INDUCED EMBRYOID FORMATION IN SUNFLOWER PROTOPLASTS

SummaryAgarose 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 μM RGD heptapeptide 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 RGD-binding proteins connected with microtubules, determining asymmetric division of the cell and polarized development.

Cytolocalization of ion-channel antagonist binding sites in sunflower protoplasts during the early steps of culture

SummaryA fluorescently labeled phenylalkylamine (PAA), DM-Bodipy PAA, was used as a probe for in vivo labeling of PAA binding sites in sunflower hypocotyl protoplasts in culture. Verapamil, a PAA known as a calcium channel antagonist in plants, lowers the division rate of sunflower protoplasts in culture. The binding specificity of DM-Bodipy PAA was established at various culture times by competition experiments with (−)bepridil. Studies on the Cytolocalization of DM-Bodipy PAA binding sites by confocal imaging showed that in freshly isolated protoplasts PAA receptors were organized into clusters uniformly distributed over the cell surface. During protoplast culture, the fluorescence labeling pattern evolved from peripheral to cytoplasmic. After a few days of culture, PAA binding sites were present inside the cell, along cytoplasmic strands, on the membrane of vesicles and vacuoles, and were highly concentrated around the nucleus. After protoplast division, the labeling was mainly restricted to a zone close to the new cell wall. On symmetrical division, binding sites were uniformly distributed on both sides of the new cell wall. With asymmetrical division, binding sites were concentrated in a ring surrounding the new cell plate.

A new tool for plant cell biology: in vivo antibody uptake in plant protoplasts

We report on the in vivo uptake of antibodies into plant protoplasts. When protoplasts of sunflower, Arabidopsis or tobacco were incubated in vivo with an antibody, this antibody was detected by immunofluorescence in the cytoplasm and/or the nucleus, depending on the location of the target protein. Furthermore, when protoplasts were cultured in the presence of antibodies, specific effects were observed. Incubation with antibodies raised against p34cdc2 led to a strong inhibition of the division rate, and a decrease in the average DNA content of protoplasts. With antibodies against HaWLIM1, a LIM domain protein of the CRP type, a negative effect on actin organisation was observed. We conclude that antibodies can penetrate plant protoplasts in vivo, and thus may be used as powerful tools for the study of protein function.

Agarose-induced embryoid formation in sunflower protoplasts is triggered by RGD-mediated membrane-matrix adhesion

Embedding of plant cells or protoplasts in gelling medium is widely used to improve cell viability and plating efficiency (Smidsrod and Skjak-Braek, 1990). In Arabidopsis thaliana (O’Neill and Mathias, 1993) and Helianthus annuus (Chanabe et al. 1991), protoplast inclusion in a solid matrix induces changes in division pattern and development. In sunflower, protoplasts cultured in liquid medium divide symmetrically and form loose microcolonies. In contrast, when they are embedded in agarose, most of them divide asymmetrically and display a polar organization at the onset of their development giving rise to compact embryo-like structures (Petitprez et al, 1995). These embryoids-progress to the heart-shape stage but do not develop further. Various hypotheses have been proposed to explain the effect of embedding on the fate of protoplasts: (i) limiting oxygen diffusion (Barbotin et al., 1993), (ii) simulating a cell wall, (iii) generating physical constraints that affect the stability of the cells (Asano et al., 1994). We previously showed that an increase of physical constraints such as hydrostatic pressure 0.4–1 MPa had an inhibitory effect on microtubule organization and cell wall synthesis and consequently on cell division (Barthou et al., 1997) but had no effect on division asymetry. Thus, physical constraints cannot solely explain the protoplast division pattern observed in agarose cultures.

Cinétique de croissance de la tige principale du soja (Glycine max (L.) Merr.). Modifications liées à la densité de peuplement

Summary The growth kinetics of the main stem of soybean have been investigated, using Neiders generalized logistics, for various values of stand density. For density values lower than 65 plants m- 2 the adult size of the stem was found to be independant of stand density. On the contrary, the various kinetic variables studied (Neiders function parameters, average and instantaneous growth rates and accelerations, growth durations) vary in a rather complex manner when stand density increases. From the analysis of these variations it has been deduced: (i) the existence of three density ranges, D≶35, 35≶D≶50 and D>50, corresponding each to different responses of the plants to density variations, (ii) the involvement of a balancing process in the growth rate/growth duration regulation and (iii) a negative correlation between stand density and the duration of the last phase of growth following the stage of maximal deceleration.