New insight into the mechanism of cellulose and callose biosynthesis: proteases may regulate callose biosynthesis upon wounding

Using a silver-enhanced, gold-secondary antibody immuno-location approach, we investigated the mechan­ isms for the switch from {3-l,4to {3-l,3-glucan biosynthesis upon wounding. Antibodies against {3-l,4and {3-l ,3-glucan synthases were used to locate these synthascs before and after wounding of Mung bean (Vigna radiata val' Berken) hypocotyls. Within 5 min of wounding, {3-l,4-glucan synthases which were densely localized on plasma membranes adjacent to the secondary walls at the wound site completely disappeared, and {3-l,3-glucan synthases became labeled. The immuno-location of the {3-1,3-glucan synthases in the secondary walls was in good accordance with the region where the ,8-1 ,4-glucan synthases were localized before wounding. Aniline blue was also utilized to visualize the deposition of callose upon wounding. Within 5 min of wounding, callose had accumulated in the corresponding region where the immuno-labeling or {3-l,3-glucan synthase was detected after wounding. The {3-1 ,3-glucan synthases were always detected from the sieve plate and plasmodesmata which are known to have constitutive synthesis of callose regardless of wounding. Secondary walls located distantly into the tissue away from the wound site were consistently labeled by the {3-1 ,4-glucan synthase antibody even after wounding. Immuno-blot analysis clearly shows that the levels of {3-l ,4-glucan synthase subunit Ces A decreased dramatically within 30 min, whereas the {3-l,3­ glucan synthase subunit CFLl levels increased significantly after wounding. The intensity of labeling reached a maximum at the wound site, and gradually decreased in correspondence with the distance from the wound site. When a protease inhibitor cocktail was applied upon wounding, neither the ,8-l,3-glucan synthase appeared nor callose was deposited during the first 5 min of wounding. On the other hand, {3-l,4­ glucan synthase was detected at the wound site, implying tha t activation or {3-1 ,3-glucan synthase may rely on the degradation of the ,8-l,4-glucan synthase. Our study may provide new insight into ,3-g1ucan synthesis in higher plants. IntrOduction the 'terminal complex' (TC) truly synthesize cellu­ lose (Brown and Montezinos 1976). TCs have a CelJulose and callose biosynthesis remains one of variety of shapes and forms among bacteria, the great enigmas in plant biology. Cellulose is a algae, and vascular plants, even including certain major component or the plant cell wall. It is known animals (Kimura and Itoh 1995). Circumstantial that aggregate granules of ordered particles termed evidence for a TC synthesizing cellulose is that it