Apical Spectrin Like Protein And Subapical Fine Bundles Of Actin Filaments Correlate With Plant Cell Tip Growth.

We studied the localization of spectrin-like epitopes in plant cells with antisera against spectrin from chicken and human erythrocytes. In various plant tissues labeling was predominant at the plasma membrane of elongating cells. To test a correlation of spectrin labeling with the site of growth, we labeled root hairs in various developmental stages and identified this epitope as a powerful marker for tip growth. The spectrin-like epitope localized in the vesicle-rich tip of growing hairs and was absent from full-grown hairs. Confocal laser scanning microscopy revealed distinct spots of spectrin labeling in the vesicle-rich region and at the apical plasma membrane. In animal cells, spectrins bound to actin can form extensive, flexible membrane-associated structures involved in maintaining mechanical properties of cells. Microinjection of purified polyclonal anti-spectrin in growing root hairs of Arabidopsis thaliana caused bursting of tips within seconds, suggesting a role for spectrin-like proteins in stabilizing the plasma membrane at the growing tip, where the cell wall is thin and flexible. Root hairs grow by exocytosis of Golgi vesicles at the tip. Vesicles are transported to the tip along actin filaments, but in the vesicle-rich tip we never observed bundles of actin filaments. In the subapical cytoplasm of growing root hairs, adjacent to the area with spectrin labeling, fine bundles of net-axially-oriented actin filaments (FB-actin) were dense. Non-growing hairs do not show subapical FB-actin, but thick bundles instead, and spectrin-like proteins are absent. Only in growing root hairs a steep tip-focused gradient of cytoplasmic calcium ions was observed, which correlated spatially and temporally with accumulated spectrin-like proteins and Golgi vesicles. This high cytoplasmic calcium ion concentration is thought to be a prerequisite for exocytosis. When host specific Rhizobium Nodulation factor is added to roots, root hair tip growth is reinitiated in hairs terminating growth. The reinitiation of tip growth consists of an apical swelling from which a new outgrowth emerges. We observed that these morphological changes were accompanied by reappearance of spectrin-like proteins and a calcium gradient at the site of exocytosis, while prior to outgrowth subapical FB-actin configuration was established. When actin-perturbing drugs like cytochalasin D or latrunculin A were applied, at vital concentrations, FB-actin disappeared within minutes, but simultaneously also the spectrin labeling and the vesicle-rich region disappeared. Similarly, the application of A23187 or EDTA to interfere with cytoplasmic calcium, not only dissipated the calcium gradient, but also caused the disintegration of FB-actin and disappearance of spectrin-like …