Microtubule involvement in the deposition of radial fibrillar callose arrays in stomata of the fern Asplenium nidus L.

Abstract

Aniline blue staining and callose immunolabeling revealed the deposition of significant callose quantities, in the form of fibrils, in the periclinal walls of guard cells (GCs) of stomata of the fern Asplenium nidus. The stomata that were at an early stage of differentiation displayed short callose fibrils at the junctions of the periclinal walls with the dorsal ones, which converged on the site of the future stomatal pore. In stomata being at an advanced stage of differentiation, callose fibrils were radially arranged around the stomatal pore, while in mature closed ones they were focused on the margins of the wall thickenings lining the stomatal pore. The pattern of the callose fibril organization resembled that of cellulose microfibrils in the same walls. Like the cellulose microfibrils, callose fibrils appeared coaligned with the underlying radial arrays of cortical microtubules (MTs). Moreover, the stomata treated with cellulose synthesis inhibitors (coumarin or dichlobenil) and those recovering from treatments with callose synthesis inhibitors (2-deoxy-D-glucose or tunicamycin) exhibited distinct radial callose fibril arrays. Cytochalasin B did not affect the organization of the radial callose fibril arrays. In contrast, oryzalin completely disturbed the pattern of callose deposition in the affected GCs. Therefore, the fibrillar callose orientation in the periclinal GC walls is probably controlled by MTs but not by actin filaments. The MTs seem to orient callose synthases in the plasmalemma, thus determining the fibrillar nature of callose deposits and their radial mode of arrangement. The cellulose microfibrils are not involved in the callose fibril alignment.