Structural analysis of cell walls regenerated by isolated protoplasts and plasmolyzed cells

Elaine Marie Shea, Purdue University

Abstract

Early studies comparing walls regenerated by protoplasts with walls of intact cells, found that cellulose synthesis was reduced and callose, (1-3)$\beta$-glucan, was synthesized instead. Most of the new material being synthesized was released to the extracellular medium instead of being incorporated into the wall. The polymers synthesized by protoplasts were different from those of normal cells. Highly purified wall hydrolases are now available and enable isolation of protoplasts in short periods of time. The exposure of the plasma membrane to these enzymes is reduced, and the many phenolics, salts and proteases contaminating other preparations have been eliminated. Protoplasts suffer little damage, and the cultures retain viability of at least 80%. During a 3 h incubation with $\sp{14}$C-glucose, less than 3% of the total material synthesized by freshly isolated protoplasts was callose, and 5-d-old protoplasts made less than 1% callose. (1-4)-Glucan synthesis by protoplasts was the same as in intact cells. With fluorescence microscopy it was shown that the (1-3)-glucan was associated only with dying cells. Protoplasts were deliberately damaged, and the synthesis of (1-3)-glucan increased in proportion to the number of dying cells in these cultures. The extracellular polymers (ECP) of healthy cultures as a percentage of the total synthesized decreased during culture, but the composition of neutral polymers in the ECP was the same. Initially the polymers incorporated into the protoplast walls were different from the walls of the source tissue, but by day 5 the new wall polymers were the same as polymers in normal walls. The percentage of uronic acid in the wall, determined by colorimetric assay, was nearly constant, but the amount of highly acidic polymers, determined by ion exchange chromatography, increased with age. When acidic groups are made available for cross-linking, pectins can form a stronger gel. The regenerated wall can be strengthened and prevent bursting in hypotonic medium. It is not necessary to remove the pre-existing wall in order to observe wall regeneration. Cells in hypertonic culture media, up to 800 mM sorbitol, remain plasmolyzed for 4 days. During that time, a wall forms around the protoplast within the pre-existing wall. The chemical composition of the newly synthesized polymers produced by the plasmolyzed cells was similar to the material produced by isolated protoplasts.

Degree

Ph.D.

Advisors

Carpita, Purdue University.

Subject Area

Botany

COinS