Involvement of an acid phosphatase on cell wall regeneration of tobacco protoplasts

Abstract

Tobacco protoplasts begin to regenerate their own cell walls, the major components of which are β‐glucans, soon after they are transferred into an adequate medium. During the cell wall regeneration the protoplasts secrete two isoforms of acid phosphatase (APase) in time‐dependent manner. We determined that one of the isoforms, the Brefeldin A (BFA) sensitive one, is the cell wall resident APase (WP‐II) by immunoblotting of the isoform with anti‐WP‐II antibody. We hypothesized that the WP‐II may participate in the deposition of β‐glucan microfibrils on the protoplast surface during cell wall regeneration. In order to examine this hypothesis, the protoplasts were cultivated in the cell wall regeneration medium containing the same amount of the BFA‐sensitive APase (230 µg protein) as is secreted by the observed number of protoplasts (1.4 × 105 protoplasts) per plate (30‐mm‐diameter) during a 3‐h cultivation after transfer to the cell wall regeneration medium. The addition of WP‐II to the cell wall regeneration medium stimulated the deposition of β‐glucan microfibrils on the surface of the protoplasts during cell wall regeneration. To determine the stimulative effect of the 60 kDa polypeptide of WP‐II, protoplasts were cultivated in the medium containing the amount of anti‐WP‐II IgG (230 µg protein) equivalent to the BFA‐sensitive APase. These results suggested that the 60 kDa polypeptide of WP‐II is the BFA‐sensitive APase which is responsible for the enhanced deposition of β‐glucan microfibrils on the surface of the protoplasts.