Effluxes of solutes from developing seed coats of Phaseolus vulgaris L. and Vicia faba l.: locating the effect of turgor in a coupled chemiosmotic system.

Cells lining the developing seed coats of legumes efflux photosynthates (mostly sucrose) and salts (mostly of potassium) into the apoplast for uptake by the developing embryo. These effluxes increase transiently in response to an increase in turgor in the effluxing cells. Detached coats of developing seed of P. haseolus vulgaris and Vicia faba were used to study the effects of turgor on the rates of efflux, on the membrane potential difference and on the membrane pH difference, using a number of inhibitors and agents which might affect signal cascades involving cytoplasmic calcium concentration. Effluxes were measured by measuring the concentrations of solutes of interest in solution samples placed in halves of detached seed coats, the paired halves serving as control and treated sample where appropriate. It is shown that a number of substances affect sucrose and potassium effluxes differently, and that hypo-osmotic shock depolarizes the efflux cells and acidifies the cytoplasm (in P. vulgaris). It is concluded that sucrose and potassium effluxes, although both are increased by an increase in turgor, are affected by different signal pathways. Further, it is also concluded that the signal that increases the rates of both sucrose efflux (via sucrose-proton antiport) and proton pump acts directly on the antiporter rather than on the pump. There are interesting parallels and contrasts between these processes and those in plants such as the charophyte Lamprothamnium after hypo-osmotic shock.

A simple and rapid technique for the immunofluorescence confocal microscopy of intact Arabidopsis root tips.

Visualisation of immunofluorescence labelling of Arabidopsis roots has previously been limited to single cell layers. A simple, rapid method has been devised in which the whole root can be processed to allow antibody penetration into several cell layers. When optically sectioned using confocal microscopy, cellular arrangements of microtubules, callose, calmodulin and a phosphoprotein epitope have been visualised using this technique. As the root is not physically sectioned, information regarding the three-dimensional position of individual cells in relation to each other and the tissue as a whole is retained. Using this technique, we have assessed the effect of brefeldin A on the frequency of mitotic arrays in root tip cortical and epidermal cells, and found that the occurrence of phragmoplasts increases significantly with brefeldin A treatment. This study demonstrates the possible future use of the whole root technique to assess rapidly the developmental, mutational and inhibitor-induced changes in the organisation of cellular components in Arabidopsis.