Geert Potters,
research assistant
Department of Biology, University of Antwerp,
Tine Raeymaekers, Nele Horemans
Evidence regarding a phenomenon is only as strong as the model system used. One needs only to consider the impact which Arabidopsis has on the development of plant science, by providing a relatively uncomplicated genetic model system. Arabidopsis is being used in many different fields of interest, for genetic, biochemical and physiological research and one cannot imagine plant science without the little weed any longer.
In a recent contribution to Plant Physiology, Geelen and Inzé (2001) discuss the potential of the Nicotiana tabacum L. cv. Bright Yellow-2 cell suspension (BY-2, Nagata et al. 1992) as a model to understand the physiological and molecular regulation of plant cell cycle progression. Because of its rapid and homogeneous growth and its highly synchronisable character (Nagata et al. 1992, Samuels et al. 1998), this suspension culture is more adequate for biochemical studies on the regulation of growth and cell division, than any Arabidopsis culture so far. Geelen and Inzé (2001) demonstrate this quite convincingly, by summarising the efforts being undertaken in the elucidation of the role and structure of the cytoskeleton and the phragmoplast during cell division. The BY-2 cell line has also succesfully been used as a model for the biochemical regulation of the cell cycle: different cyclins and CDKs (the ‘motor proteins’ of the cell cycle machine) have been identified in and isolated from BY-2 cells. Also, plenty of data have been gathered with these BY-2 cells, concerning the role of auxin (Redig et al. 1996), cytokinin (Laureys et al. 1998, 1999), abscisic acid and jasmonic acid (Swiatek et al. 2002), all able to intervene in (or necessary for) normal cell cycle progression.
However, it is very unfortunate that the authors did not discuss the evidence which has been obtained to date, with the BY-2 model system, on the role of ascorbate (ASC) and glutathione (GSH) in the regulation of cell division. While we strongly support the conclusions of Geelen and Inzé (2001), we feel that an even stronger case in favour of the BY-2 suspension can be built: especially the research on ASC and GSH in BY-2 cells backs the claim that this cell suspension is useful as a model system for the study of cell growth, not only on a molecular basis, but also in physiological research. To illustrate this point, we will first present an overview of the data concerning ASC, GSH and cell growth in BY- 2 cells, and on the possible interaction of ASC metabolism and hormone regulation of cell growth. On the other hand, the conditions for a cell in suspension are far away from the conditions in a normal plant, like for example, a root or a shoot meristem. A suspension cell culture may therefore not be suitable for every physiological experiment, and caution should be taken, when extrapolating results obtained with this system, to explain effects on a tissue scale. Fortunately, as we will shown below, there is also a number of experimental data, obtained on intact plants, concerning ASC metabolism and cell growth control to confirm the effects observed on BY-2 cells.
ASC and GSH influence cell division in BY-2 cells
The last decade has witnessed the discovery of several links between ASC metabolism and cell growth or cell cycle regulation. The BY-2 cell culture has proven an interesting system in this respect. For example, during the growth of a BY-2 cell culture, a transient peak in the endogenous ASC level was shown, during the period when the cells were dividing exponentially. Upon entrance in the stationary phase, the ASC concentration and redox status decreased (Kato and Esaka 1999, De Pinto et al. 1999). Several ASC-related enzymes (ASC peroxidase, DHA reductase) show an activity profile, depending upon the growth cycle stage of the BY- 2 culture (De Pinto et al. 2000). Moreover, Kato and Esaka (1999) demonstrated a transient peak in the DHA concentration, concomitant with a decrease in the ASC/DHA ratio during M-phase. This increase also correlated with a temporary increase in ASC oxidase expression during G2 and M-phase (Kato and Esaka 1999). Other enzymes of the ASC metabolism show a cell cycle phase-dependent change in activity as well.
Upon the addition of extra DHA (1 mM in the BY-2 culture medium) during the G1 phase, the progression of the cell cycle was temporarily halted (Potters et al. 2000). As far as the mechanism of this block is concerned, it was shown that the incoming DHA is quickly reduced by the BY -2 cells, and that the compound responsible for the DHA reduction will probably provide the connection with the cell cycle. The identification thereof is still taking place, though different hypotheses on the identity of the DHA reductant exist (De Pinto et al. 1999, Potters et al. 2000).
And what about the other important antioxidant – GSH ? This compound was linked to cell cycle regulation upon the discovery of a GSH-dependent developmental pathway, necessary for cell division initiation and maintenance in the root meristem. Apparently the ROOT MERISTEMLESS1 (RML1) gene actually codes for the γ-glutamylcysteine synthase, the rate -determining step in GSH biosynthesis (Noctor et al. 1998, May et al. 1998). Plants homozygous for a mutation in RML1, had only 3% of the wild type GSH level. These results were confirmed with the BY-2 cell culture: in a GSH depleted BY-2 culture blocked cell cycle activity at the G1/S transition. Two different A-type cyclins, involved in this transition, were also downregulated (Vernoux et al. 2000).
Two conclusions can be drawn from these data. Firstly, ASC metabolism is tightly tied into cell growth and cell division. Secondly, intracellular levels of DHA are under the control of the cell cycle, through the expression of ASC oxidase, which is, surprisingly, an apoplastic enzyme. This internal elevation of DHA levels may be crucial in the decision whether to start another round of the plant cell cycle, or not. An elevated DHA level may be interpreted as a stress signal, and be designed to halt energy-consuming processes, like cell division, energy that is better spent on defence. Elevated DHA levels (or an elevated DHA production) are typically associated with stress (Gossett et al. 1994, Luwe et al. 1993, Takahama 1993, 1994). Under normal conditions, the influx of DHA from the apoplast will subside as soon as the ASC oxidase activity (in G2 and M phase) decreases, and the DHA level in the cytoplasm will return to normal. The occurrence of stress conditions will keep the internal DHA concentrations elevated (irrespective of the ASC oxidase activity), and thus influence the decision whether to enter another round of the cell cycle, or not.
BY-2, ASC and auxin: a trio for cell elongation research ?
In the light of its easily obtained synchrony with respect to cell cycle progression, it is readily forgotten that a specific treatment with phytohormones will force a BY-2 cell culture into synchronous elongation (for details see Hasezawa and Syono 1983). The hormone treatment apparently changes the culture from dividing to elongating. This should not come as a surprise. The effect of hormones, and particularly auxin, on cell elongation has been studied for decades. Every textbook on plant physiology and biochemistry discusses the auxin-induced elongation, and mentions several arguments à charge and à décharge in the process. However, during the last decade, another player has entered the elongation field – ASC. For example, upon synchronisation of BY-2 elongation, Kato and Esaka (1999) showed that this phase of extreme elongation of the BY-2 cells is accompanied by a tenfold increase of ASC oxidase activity in the culture medium. This is all the more interesting, since the question about the role of the ASC oxidase in plants had never been solved. Although the structure and the biochemical characteristics of this enzyme are extensively studied, the function of the enzyme has not been clarified yet. Yet, the observation of ASC oxidase activity correlating with cell expansion (Kato and Esaka 1999, Kato and Esaka 2000) may just as well provide the proper answer. Indeed, during the last decade, a model has been formulated concerning the rather peculiar stimulation of cell elongation by MDHA (González-Reyes et al. 1998). It has been assumed that MDHA is able to invoke an energisation of the plasma membrane, an activation of the plasma membrane ATPase, which will then enhance cell wall acidification. Transmembrane electron transport in general stimulates H+-ATPase activity and leads to cell wall loosening (González-Reyes et al. 1994). Recent evidence suggests that MDHA could act as both electron donor and acceptor in transmembrane electron transport (Asard et al. 1995). MDHA also causes an increased vacuolisation, a process which drives cell elongation even more (review, González-Reyes et al. 1998). A tight regulation of the ASC oxidase activity might therefore just as well serve to control the onset or the progression of cell elongation (Kato and Esaka 1999). Construction of a transgenic BY-2 cell line, overexpressing ASC oxidase, confirmed this theory: protoplasts isolated from the transgenic lines elongated more rapidly after a proper hormone treatment than those of control lines (Kato and Esaka 2000). In addition, the expression of the ASC oxidase gene is regulated by auxin (Esaka et al. 1992, Kerk and Feldman 1995), but also, the auxin concentration itself may be modified by ASC oxidase (Kerk et al. 2000). It would be very interesting how far this connection between auxin and ASC metabolism stretches.
Validity of the BY-2 cell suspension for research on ASC and cell cycle
As demonstrated above, the BY-2 culture is quite advantageous for the study of cell cycle progression and cell elongation. However, similar results have been obtained in experiments with intact plants. For example, DHA was proven to block entry into S phase in onion root tips (De Cabo et al. 1993). Kerk and Feldman (1995) explained the existence and maintenance of a quiescent centre in root tips by pointing out that ASC oxidase activity is quite high in these cells. The concomitantly higher DHA concentration in these cells could be the agent blocking cell cycle progression (in G1). The experiments with DHA addition were also repeated by Paciolla et al. (2001). Even in root tips of lupine or onion, mitotic activity decreased, as had been observed in BY-2 cells. A more thorough review on ASC, GSH and cell growth will appear shortly in Plant Physiology and Biochemistry. Nevertheless, even this selection of data should be sufficient for a comparison between BY-2 cells and intact plants. In both systems, DHA has been shown to block the cell cycle temporarily, and apparently, the DHA effect is somehow linked to the G1/S transition. Moreover, as discussed above, observations on the effect of ASC and ASC metabolism on cell elongation, both in BY-2 cells and in intact plants, apparently corroborate each other. The BY-2 cell system is therefore an appropriate system to study both types of plant cell growth, by division and by expansion. This makes the BY-2 cell line not only a useful tool for cell cycle research, but for cell growth in general.
The numerous observations pertaining to the correlation between ASC metabolism in intact plants, different tissues, and the BY-2 cell culture point towards a similar involvement of ASC in the regulation of growth in all cases. We feel that it is therefore sufficiently demonstrated that the effects observed in BY-2 cells are physiologically relevant, and any hypothesis on a physiological function of the observed phenomena will probably sustain the scrutiny of testing in intact plants.
Drawbacks of the current methodologies : a bright solution ?
A common critique on the experiments in which DHA is added to the culture medium, is that the observed effects on the cell cycle, are not specifically due to the impact of DHA on cell metabolism. Rather, the addition of 1 mM DHA may have unspecific toxic effects. It has been stated before (Bianchi and Rose 1986) that a DHA build-up may damage membranes and proteins. On the other hand, viability was never compromised by the amount of DHA in our experiments (De Pinto et al. 1999, Potters et al. 2000), and when allowed to continue growing after completion of an experiment, normal growth was resumed (De Pinto et al. 1999). Concentrations up to 1 mM ASC are regularly found in plant tissues ; Foyer and Lelandais (1996) report even values from 1 to 5 mM for the apoplastic ASC concentration. The normal concentration found in normal BY-2 medium, however, did not surpass 20 µM, and the amount of ASC was completely oxidised (Potters et al. 2000). Luckily, the problem may be avoided in the future. BY-2 cells are readily transformed (Geelen and Inzé 2001). With the molecular data concerning ASC and GSH biosynthesis and regeneration becoming more clear (Conklin et al. 1996, Urano et al. 2000, Asard et al. 2001, Smirnoff et al. 2001, Xiang et al. 2001), the BY-2 system should provide plenty of opportunities to devise well targeted experiments, tackling only one component of the metabolism of ASC or GSH at a time, and observing specific effects. Tabata et al. (2000) have already created a transgenic BY-2 line, producing less ASC than their wild type counterparts. These cells show an abnormal (slower) cell cycle progression. Application of this strategy to other enzymes will be very useful to study the intertwining of ASC metabolism and cell growth, without having to resort exclusively to the cruder experiments of today.
Conclusion
Two conclusions can be drawn here. First, the data summarised here provide a valid extension of the argumentation of Geelen and Inzé (2001). Indeed, the BY-2 culture is an easily handled model system, which has been found physiologically relevant, up to now. Because of its peculiar physiological condition, comparison with an intact plant model system, however, should not be left out of future research. Arabidopsis is more than sufficient to check genetic data, tobacco or tomato plants should be used as a model in more physiological studies. Secondly, when it comes to a description of the events surrounding the regulation of cell growth (division and elongation), redox compounds have been demonstrated to be as equal a team player as the far more frequently mentioned hormones. Both types of physiologically relevant molecules have their own function, and it would not be surprising if many interconnecting pathways would be discovered. In fact, a picture of the link between auxin and ASC is slowly emerging, and the existing data are clamouring for further experimentation, focusing on both redox compounds and plant hormones. The BY-2 future indeed looks bright!
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