Ascorbate and dehydroascorbate influence cell cycle progression in a tobacco cell suspension.

In addition to its well-known antioxidant properties, ascorbate (ASC) is capable of influencing normal cell cycle progression in plants. In this work we demonstrate that the oxidized molecule dehydroascorbate (DHA) is the active redox form in this respect. Our results indicate that the reduction of DHA might constitute the first step leading to this effect. During the last few years, evidence has already been accumulating, indicating the role of ASC in the regulation of cell division. For example, De Cabo et al. (1993) demonstrated a shortening of the G1 phase in dividing onion root meristem cells due to the action of monodehydroascorbate. Kerk and Feldman (1995) were able to establish a direct correlation between ASC redox status and cell proliferation rates in the maize (Zea mays) quiescent center. Also, during the growth cycle of a tobacco (Nicotiana tabacum L. cv Bright Yellow-2 [BY-2]) cell suspension, a significant decrease in the endogenous ASC level was shown, accompanied by an overall decrease in redox status (De Pinto et al., 1999; Kato and Esaka, 1999). Kato and Esaka (1999) provided further evidence pointing at a possible ASC-mediated redox control of the cell cycle by demonstrating a transient peak in the DHA concentration, with a concomitant decrease in the ASC to DHA ratio during M phase. This increase also correlated with a temporary increase in ASCoxidase expression. These results suggested the hypothesis that intracellular levels of DHA might be controlled by the cell cycle through the expression of ASC-oxidase. In addition the decrease in DHA could also constitute a necessary and positive signal for the cell to proceed into S phase (Kato and Esaka, 1999). De Pinto et al. (1999) also suggested a role for DHA as a specific redox link between the apoplast and the cytoplasm. Because ASC is the major antioxidant in the apoplast, it is a likely candidate to play a role in the signaling from the apoplast to the cytoplasm related to the oxidative properties of a possibly stressful environment. The presence of a DHA transporter has recently been demonstrated at the plasma membrane of higher plant cells (Horemans et al., 2000). This transporter assists in the uptake into the cell of apoplastic DHA, resulting from the consumption of apoplastic ASC in a variety of different possible oxidative reactions; therefore, the transporter might relay the apoplastic redox status to the interior of the cell. The addition of external DHA is likely to affect the internal redox balances, possibly through its glutathione-dependent reduction. In this respect Reichheld et al. (1999) suggested the existence of an oxidative stress checkpoint pathway that controls cell cycle progression in environmental stress conditions and is seemingly responsive to one or more redoxsensing systems. For example, menadione (known to cause oxidative stress by generating oxygen radicals) has been shown to impair the G1/S phase transition in BY-2 cells (Reichheld et al., 1999). Thus, the ASC molecule is gradually considered not only an essential antioxidant, but it also plays a key role in plant cell signaling. The literature discussed so far led to the hypothesis that induction of a shift in the intracellular ASC to DHA ratio should influence cell cycle progression. Here we describe the progression of the cell cycle after addition of ASC and DHA to a synchronized BY-2 cell suspension culture (Nagata et al., 1992). The results show an inhibition of cell cycle progression and provide a basis for the development of future experiments to uncover the links between oxidative stress, cellular redox status, and the control of cell division.