Aspects of plant cell growth and the actin cytoskeleton : lessons from root hairs

The main topic the thesis addresses is the role of the actin cytoskeleton in the growth process of plant cells. Plant growth implies a combination of cell division and cell expansion. The cytoskeleton, which exists of microtubules and actin filaments, plays a major role in both processes. Before cell growth takes place, a new cell is formed by cell division. The orientation of the division plane most often predicts the orientation of cell expansion, and a correct positioning of the division plane is therefore important for plant morphogenesis. During most stages of cell division microtubules and actin filaments have a similar configuration.In Chapter 1 (De Ruijter <em>et al.</em> , 1997, <em>Acta Bot. Neerl</em> . 46: 279-290) the cytoskeleton of microtubules has been visualized during all stages of cell division for long and short root tip cells of broad bean ( <em>Vicia faba</em> L.). In all cells the preprophase band of microtubules was positioned in the midplane of the cell, and perpendicular to the long axis of the root. It was observed that the spindle axis in short cells increasingly tilted, from meta- to anaphase, giving rise to oblique cell plates <strong>.</strong> It appears that this is caused by spatial constraints. During late-telophase, cell plates first rotated towards the transversal plane before they fused with the parental wall at the site of the earlier preprophase band. When cell division is completed, cells grow.Plant cell growth is the insertion of Golgi vesicles into the plasma membrane and the delivery of their content into the existing wall. If this wall is flexible and under turgor pressure, the membrane becomes larger and the wall expands. The basic principles of plant cell growth can best be studied in cells where this growth process takes place abundantly, that is in the tip of tip-growing cells of higher plants, such as root hairs and pollen tubes. In Chapter 2 (De Ruijter <em>et al</em> ., 1998, <em>Plant J.</em> 13: 341-350) characteristics for cell tip growth are being reported, studied by comparison of developmental stages of root hairs of vetch ( <em>Vicia sativa L</em> .), from their emergence to their maturity. It is further shown that lipochito-oligosaccharides (LCOs), well-characterized molecules that are excreted by bacteria, reinitiate cell tip growth in hairs that are terminating growth. Tip growth and the site of growth re-initiation correlates with the presence of a steep cytoplasmic calcium gradient at the plasma membrane. Furthermore, it was found that a spectrin-like protein is a good marker for tip growth, and co-localizes with the vesicle rich region, which is known to be present at the tip.Immunolocalization of this spectrin-like protein in plants was extended to a variety of growing cells and shows, in Chapter 3 (De Ruijter and Emons, 1993, <em>Cell Biol. Int.</em> 17: 169-182), that this protein is especially present in young growing cells. Molecular weight and iso-electric point determination, by means of immuno-blotting identified the plant spectrin-like protein. The anti-spectrin antibody also labels nuclei, which is further investigated in Chapter 4.To analyze the presence and localization of nuclear spectrin-like proteins, various plant tissues and isolated pea nuclei were labeled. The data presented in Chapter 4, show that the spectrin-like proteins are distributed in a speckled pattern and occasionally in tracks. The extraction procedures used indicate that the spectrin-like protein is part of the nuclear matrix in which it may be a stabilizing factor.In Chapter 5 (Miller, De Ruijter <em>et al.,</em> 1999, <em>Plant J.</em> 17: 141-154) the actin cytoskeleton of vetch root hairs at their initiation and during their development is described. Actin filament bundles are the dynamic backbone of the cytoplasmic strands. Growing hairs show dense sub-apical fine bundles of actin filaments (FB-actin) and the very tip is devoid of actin filament bundles, whereas full-grown hairs have actin bundles looping through the tip. Similar actin configurations were obtained when root hairs were freeze substituted and immunolabeled with anti-actin, or chemically fixed by an improved method and stained with fluorescent phalloidin. Since LCOs had been shown to reinitiate root hair growth (Chapter 2), this signal molecule was used to study the actin cytoskeleton during growth reinitiation. Manipulation of the actin cytoskeleton with the actin filament capping drug cytochalasin D inhibited polar growth. However, root hair initiation and swelling after LCO application were not affected. We concluded that elongating FB-actin is another characteristic for tip growth.Indeed, LCOs altered the configuration of the actin cytoskeleton, which was studied in Chapter 6 (De Ruijter <em>et al.</em> , 1999, <em>MPMI</em> 12: 829-832). The density of sub-apical actin filament bundles increased within 3-15 minutes after the application of LCOs. By a quantitative approach we were able to define the minimal FB-actin density and minimal length, of the area with the FB-actin, needed for growth. Only in hairs in which FB-actin exceeded these values, tip growth was sustained or resumed. The rapid response of actin filaments indicates a role for the actin cytoskeleton in signal transduction cascades.Such a dynamic actin cytoskeleton must be regulated. Part of this regulation will be done by actin binding proteins. Therefore, our limited knowledge of actin binding proteins in plant cells is reviewed in Chapter 7 (De Ruijter and Emons, 1999, <em>Plant Biology</em> 1: 26-35) of the thesis.Chapter 8 summarizes the characteristics for growth in tip-growing cells and extrapolates them to cells that expand isodiametrically or predominantly along one length axis. We conclude that tip growing cells, like root hairs, shed light on basic principles of plant growth, and provide a system to monitor the effect of signal molecules on cell growth.