Chapter 7. The Vacuole7.1. Discovery of the vacuoleMost plant cells contain a conspicuous central region that appears empty in the light microscope (von Mohl, 1852). This region, which includes a transparent, or rarely colored, watery substance, known as the cell sap is called the vacuole, a term which comes from the Latin word for empty. The large central vacuole can take up approximately 95% of the protoplasmic space although in typical higher plant cells, it takes up approximately 60% (Figure 7-1). A large central vacuole is not limited to plant cells. According to Bensley (1951), a large central vacuole is also found in the cells of the flagellate Noctiluca, the ciliate Trachelius, the ectoderm and endoderm of the Coelenterates, and in the Heliozoa.Vacuoles were first observed in protozoa. The contractile vacuoles or “stars” of many protozoa were seen by Spallanzani (1776) although he mis-took them for respiratory organs (see Zirkle, 1937). These “stars” were named vacuoles by Dujardin (1841). Although the optically structureless cell sap had been observed by botanists for years, the term vacuole was first ap-plied to plant cells by Schleiden in 1842 when he distinguished the vacuole from the rest of the protoplasm (Zirkle, 1937).The cell sap is surrounded by a membrane as determined from osmotic studies done by Hugo de Vries on Tradescantia epidermal cells and many other cell types (1884a,b,1885,1888a,b). In these studies he noticed that the cell walls bulged when the cells were placed in pure water. As he increased the concentration of solutes in the external solution, the walls relaxed, and, at higher concentrations of solutes, he observed that the violet colored vacuole shrank. de Vries concluded that a membrane must surround the cell sap in order for the vacuole to behave as an osmometer. He coined the term tonoplast to designate the membrane that surrounded the cell sap. The tonoplast was so named because he thought that it was the regulator of turgor--or tonicity in the cell (de Vries, 1910). He mistakenly believed that the tonoplast was differentially permeable but the plasma membrane was not and consequently, only the tonoplast regulated turgor. 221de Vries (1885) also thought that the tonoplast was an autonomous self-replicating particle in the cell. However Wilhelm Pfeffer (1900-1906) showed that vacuoles are not autonomous but form de novo during phagocytosis. Nowadays it is possible to observe vacuoles develop in evacuolated (Hörtensteiner et al., 1992) or vacuole-less protoplasts (Davies etal., 1996). Since the tonoplast is neither self-replicating nor the primary site ofturgor regulation, I will use the term vacuolar membrane to denote the differentially permeable membrane that surrounds the cell sap as suggested byPfeffer (1886).7.2. Structure, biogenesis and dynamic aspects of vacuolesAlthough a few meristematic cells, including the apical cell in Osmunda and Lunularia as well as the cambial initials in higher plants have prominent vacuoles (Bailey, 1930; Sharp 1934), the vacuole is inconspicuous in most meristematic cells (Porter and Machado, 1960). The development of the vacuole can be followed in the light microscope. For example, Pensa (see Guilliermond, 1941) looked at the development of the vacuolar system in the cells of the teeth of young, living rose leaflets (Figure 7-2). The vacuolar systems in these cells are easy to observe since the vacuoles are filled with anthocyanin. In the youngest cells at the tip, the vacuoles appear as numerous,tiny filamentous elements. In slightly older cells, these filamentous elements appear to swell. Eventually, in the mature cells at the base, the swollen elements fuse into larger vacuoles and eventually form a large central vacuole.Dangeard (1919) gave the name vacuome or vacuolar system to all the vacuoles contained in the cell during all its phases of development. A similar vacuolar development can be seen in maturing barley root cells stained with neutral red, a vital stain that is preferentially taken up into acidic compartments (Figure 7-3). Other good examples of vacuolar development include the epidermal cells of young leaves and the hairs on the sepals of Iris germanica, the glandular hairs on the leaflets of walnut, and the leaves of Anagallis arvensis. By contrast, the vacuolar system of Elodea canadensis never goes through a filamentous stage, but starts as small spherical vacuoles that later fuse into a large central vacuole (Guilliermond, 1941). A more recent study of vacuolar development has been done using the autofluorescent vacuole found in the developing guard cells of Allium cepa byPalevitz and O'Kane (1981) and Palevitz et al. (1981). They find that the vacuoles of young guard mother cells are globular. As the guard mother cells 222develop, the vacuole is transformed into a reticulum of interlinked tubules andsmall chambers. The tubules are approximately 100-500 nm in diameter. In the guard mother cell, the network continually undergoes changes in shape and remains reticulate during the division that gives rise to the two guard cells. The reticulate networks persist through the early stages of guard cell differentiation and then they are transformed into two large globular vacuoles, one in each guard cell (Figure 7-4). The dynamics of the vacuolar compartment has also been confirmed using various vacuolar proteins fused to GFP (Flückiger et al., 2003; Hicks et al., 2004; Zouhar et al., 2004).The developmental pattern seen in vacuoles can be reversed under certain physiological conditions. For example, Charles Darwin (1897) noticed that the vacuole of the tentacle of the carnivorous plant Drosera rotundifolia, which is filled with anthocyanin, appears to break up after the leaf is stimulated by an insect (Actually Darwin misidentified the vacuole as protoplasm). The cells of the tentacles contain a single central anthocyanin filled vacuole. At the moment of stimulation, the vacuole fragments into filamentous vacuoles. Immediately after stimulation, the filamentous vacuoles fuse to form a large central vacuole and the cell returns to its initial state (de Vries, 1886; Guilliermond, 1941; Lloyd, 1942; Juniper et al., 1989).The minimal requirement for the formation of vacuoles is the synthesis ofa vacuolar membrane that contains the transporters necessary to increase theosmotic pressure of the lumen. The increase in the osmotic pressure will allow the newly formed