In the early response of Arabidopsis roots to ethylene, cell elongation is up- and down-regulated and uncoupled from differentiation.

Slight changes in the concentration of ethylene in the environment modulate the elongation of target cells in the root epidermis of Arabidopsis. The response is immediate, concentration dependent, and reversible on root base but irreversible on cell base, whereas cell differentiation is not affected. We suggest that in natura ethylene is a means of fine and fast tuning of root elongation. In Arabidopsis, root elongation is reduced in a concentration-dependent way and radial expansion is stimulated when plants are exposed to ethylene or 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene (Jackson, 1991; Dolan, 1997; Smalle and Van Der Straeten, 1997). In addition, root hair growth is promoted by ACC. Anatomical and clonal analysis revealed that the root epidermis is composed of two types of cells occurring in parallel files along the root: trichoblasts and atrichoblasts (Dolan et al., 1993, 1994; Galway et al., 1994; Schneider et al., 1997). Ethylene can also affect differentiation patterns by inducing ectopic root hair formation in atrichoblast cell files (Tanimoto et al., 1995; Masucci and Schiefelbein, 1996; Pitts et al., 1998). However, the precise mechanisms governing these ethyleneinduced changes in cell development are not fully understood. The effect of ethylene on root development usually is scored after long incubation times (many hours to days) of the plants, often using high concentrations of ethylene or ACC. Such reports can be useful from the standpoint of a bioassay, but have much less potential in supporting theories pertaining to root development. Only a limited number of reports refer to short-term responses of roots (Jackson et al., 1981; Whalen and Feldman, 1988). We analyzed the effect of ethylene on the primary root of Arabidopsis, focusing on the very early responses (within minutes) of the epidermis. For this purpose, we used a confocal microscope to monitor minute changes in the elongation of individual cells in intact growing roots. In Arabidopsis roots, mature cell length normally was used to analyze the effect of different mutations or hormone-treated seedlings on a cellular basis (Masucci and Schiefelbein, 1996; Rahman et al., 2000), but this parameter is not suitable to record fast responses and fine dose-dependent regulation of cell elongation. In wild-type roots, grown on hormone-free Murashige and Skoog medium, fast-cell elongation occurs between 250 and 950 mm from the root tip (Beemster and Baskin, 1998; Mullen et al., 1998). Within this zone, the cells elongate along the root growth axis from 15 to 130 mm. The formation of a root hair bulge is often taken as a marker for the switch from elongation to differentiation. Epidermal cells do not cease elongation immediately, however. They elongate further during 1 to 1.5 h until they reach the mature length of 150 to 200 mm. Mature cell size can thus only be evaluated in single still images taken far from the root tip; this implies the loss of information on early responses to ethylene. Therefore, we introduce the length of the first epidermal cell with visible root hair bulge (LEH), counting from the root tip, as a new parameter of root development. It marks the onset of differentiation and the end of rapid elongation (Fig. 1). The movie (http://www.uia. ac.be/bio/fymo/lejie/root.html) illustrates the rapid elongation of cells shorter than LEH, and the slow elongation in cells longer than LEH. Root hair initiation or bulging is strictly regulated in time and space and happens every 27 min in each trichoblast cell line. As shown in the movie, LEH can be easily recognized and measured. In seedlings grown on a medium containing ACC, the value of LEH is reduced with a dose-response ratio similar to that of total root length (Fig. 2). Therefore, a specific level of ethylene (or ACC) defines a specific LEH, the cell size reached at the onset of differentiation. When a growing plant root is suddenly exposed to ethylene (1 mL L), the effect on cell elongation is dual. In the elongation zone the epidermis cells having a length greater than or equal to the LEH corresponding to the given ethylene concentration immediately stop their elongation. Cells closer to the root tip with a length less than LEH elongate further until 1 This work was supported by the Fund for Scientific ResearchFlanders (FWO, Belgium; grant nos. G.0034.97 and G.0281.98). * Corresponding author; e-mail [email protected]; fax 32– 3– 820 –2271.