Only a small number of papers have investigated anthocyanin accumulation in individual cultured plant cells through image analysis, although interesting attempts to investigate these complex systems have been made by counting pigmented and nonpigmented protoplasts derived from cell suspension using an optical microscope. With this latter technique, it has been shown that high pH in the growth medium increases the anthocyanin content of strawberry cultures (biochemically determined) without changing the percentage of pigmented cells, suggesting that anthocyanin accumulates inside pigmented cells (Zhang & Furusaki, 1997). However, this approach relies on the subjective perception of the operator. An objective description of cell pigmentation can be performed using digital image analysis with specific colour models.
Two main colour models have been used, namely RGB (Miyanaga et al., 2000a,b) and HSI (Smith et al., 1995). The RGB model describes colours as three selected clusters of light (red, green and blue). Miyanaga et al. (2000a) described the colour characteristics of anthocyanins as having red component values that were much larger than the green or blue ones. It was thus proposed that the difference between the red and green component be used as an index for anthocyanin accumulation. Using this method, the above authors demonstrated a correlation between the pigmented cell ratio and the average anthocyanin content in strawberry cultures (Miyanaga et al., 2000a). Moreover, it was found that the anthocyanin content of individual cells underwent sharp variations during time (Miyanaga et al., 2000b).
The HSI model is more intuitive than RGB, because it describes colours as they are perceived by the human eye. Hue is the pure colour, described by a number from 0 (red) to 360 degrees (red), passing from yellow, green, cyan, blue and magenta. Saturation is the measure of the degree to which pure colour is diluted by white light, higher amounts of white corresponding to lower saturation, and gives an estimation of the purity of the colour. The intensity value estimates the darkness of the colour, with higher amount of black corresponding to lower intensity. The values of S and I are usually expressed in 0–1 range. In our system the three HSI values are all converted into 255 channel values.
In transmitted light microscopic images of individual pigmented cells, S and I values are not completely independent, in accordance with the observations of Smith et al. (1995). This is due to the fact that the colour of the cell depends on the amount of pigment and on the interaction between the pigmented cell and the incident light of the microscope. Highly pigmented cells, for example, absorb and reflect more light, and this results in less light arriving at the eye or the camera, and thus in more saturated (less amount of white) and less intense (greater amount of black) images.
Smith et al. (1995) used the HSI colour model to analyse Ajuga anthocyanin-producing cell cultures. Hue values were used to segment the total image pixels into pigmented (H > 240) and nonpigmented pixels, while saturation values were used to further segment pigmented pixels in lowly (0 < S > 0.1), medium (0.1 < S > 0.2) and highly pigmented (0.2 < S > 1) ones.
We used the HSI model and biparametric HS plots of pigmented and nonpigmented cells in order to evaluate the accumulation of anthocyanins in carrot cell cultures. Our observations suggested that the hue value alone is not sufficient to discriminate between white and pigmented cells, since a certain number of cells of the pigmented cultures showed the same H-value range of cells from a nonpigmented culture, but with higher saturation values. The analysis of selected cells perceived as low-pigmented by visual inspection confirmed that these cells have the same hue values of white cells, but higher saturation values. Segmentation based on HS two-dimensional plots instead of an one-dimensional H-value allowed us to identify two different populations of pigmented cells: one that has lower and more scattered hue values (included in R2 region), and the other with higher hue values and higher and more scattered saturation values (included in R3 region). According to the analysis of visually selected pigmented cells, the latter cell population includes more strongly pigmented cells.
Since saturation distribution within the individual regions roughly correlated with the pigmentation level perceived visually (Fig. 2c), the average saturation value of each region was used to evaluate the accumulation/loss of anthocyanins within the population of low-pigmented and high-pigmented cells (Fig. 3b).
Effect of the reduced batch volume on cell growth and anthocyanin content
The time-dependent effects on growth and anthocyanin content and yield were determined in anthocyanin-producing cells originated from the same batch and cultured in two different experimental conditions, namely 50-ml batch culture in 250-ml flasks, which is also the standard growing condition for this cell line, and 20-ml batch culture in 250-ml flasks. Cells in the latter condition were characterized by a much higher anthocyanin content and yield during a normal culture cycle of 14 d.
The reduction of the batch volume is known to increase gaseous exchange and therefore the oxygen supply, owing to the higher volumetric oxygen transfer, which depends on the surface/volume ratio and on the mixing intensity (Gao & Lee, 1992). This latter parameter, in turn, is higher for smaller volumes because of the increase of the empty space in the flasks for liquid movement during agitation. Therefore, cells in the 20-ml batch culture should have a higher oxygen supply and a higher mechanical stress compared with 50 ml batch cultures.
An increase in oxygen concentration has been reported to increase the growth rate in many plant cell cultures, including tobacco (Gao & Lee, 1992), Catharanthus roseus (Tate & Payne, 1991) and carrot (Jay et al., 1992).
The effect of fluid mixing on cell growth is, however, more complex, since poor mixing causes oxygen limitation with consequent limitations on growth (Zhong et al., 2002), while mixing that is too vigorous causes hydrodynamic stress that can result in cell damage and a decrease in biomass production (Ho et al., 1995). In general, mechanical stress in cell cultures can generate oxidative stress, as described, for example, in Taxus cuspidata (Han & Yuan, 2004) and soybean cell cultures (Legendre et al., 1993) even in the absence of apparent mechanical cell damage.
In our system, the reduction of the batch culture volume led to an increased growth rate during phase 3 in 20-ml batch cultures, which reached a growth plateau at earlier times. According to literature data, this could be an effect of the improved oxygen supply.
However, the analysis of individual cells revealed a much more complex situation: growth of the high-pigmented cell subpopulation was higher in the 20 ml batch culture up to day 14, which had positive effects on the anthocyanin yield. By contrast, the growth rate of nonpigmented cells during phase 3 increased. These events are expected to affect the anthocyanin yield in an opposite manner.
The analysis of the relative abundance (%) of the three types of cells during the same phase also suggested that at least part of the new, nonpigmented cells are derived from the low-pigmented subpopulation. In vivo imaging showed that in the late phases of culture (final 5 d) the shift of cells from pigmented to nonpigmented subpopulations is mainly (more than 90%) the result of cell death. The experimental conditions during in vivo imaging were quite different with respect to the time-course experiments in which cells were growing in liquid batch in shaken flasks. For in vivo imaging, cells were plated on a thin layer of liquid medium over solid medium within a large dish. Because of these experimental conditions, colour analysis could not be performed, and cells were visually classified as white, low-pigmented and high-pigmented. Therefore, in vivo imaging allowed only a rough estimation of the possibility and frequency of these events. Nevertheless, the above observations suggest that the reduction of the batch volume could cause cell death of low-pigmented and nonpigmented cells, while specifically improving the growth of high-pigmented cells.
The increase in the oxygen concentration, which in our system was achieved through the volume variation of the batch cultures, has been reported to increase the production of secondary metabolites such as ajmalicine from Catharanthus roseus (Schlatmann et al., 1994), berberine from Thalictrum minus (Kobayashi et al., 1989) and anthocyanins from Perilla frutescens (Zhong et al., 1993).
In our system, the effect of the culture volume on anthocyanin content appeared complex. At the time of subculture (14 d), the anthocyanin content of the 50 ml batch was still increasing. The very early anthocyanin content peak in both 20-ml and 50-ml batch cultures could be seen as a sort of continuation of the anthocyanin content peak of this late phase, which is characterized by an abundant biomass still growing and accumulating anthocyanins. Alternatively, the initial anthocyanin peak could be a specific effect of the new environmental conditions following the reinitiation of the culture cycle, characterized by fewer cells in fresh medium. During phase 1, other parameters, such as the average S-values of the different cell subpopulations and their relative abundance, also showed that same trends were present at day 14 of a 50-ml batch culture, indicating the former hypothesis is more plausible. The fact that conditioned medium and abundant biomass, rather than fresh medium, is generally described as promoting secondary metabolites production (Lee & Shuler, 2000; Zhang et al., 2002) supports the same conclusion.
Therefore, the anthocyanin content during phase 1 seemed to be strongly affected by the productivity and the physiological state of the inoculated cells.
The enhancement in both the content and yield of anthocyanin during phases 2 and 3 in the 20 ml batch culture compared with 50 ml batch culture could be ascribed to two synergistic events. These include the enhanced high-pigmented cell growth and the enhanced accumulation of pigments into high-pigmented cells. A third important event, the enhancement of nonpigmented cell growth during phase 3, negatively affects the anthocyanin content and yield.
Our observations might be explained by considering that a decreased culture volume leads to an increased oxygen supply, with a subsequent positive effect on cell growth. However, this condition may cause some type of cell stress, for example mechanical stress, which may result in oxidative stress. Considering the above, anthocyanins have the ability to protect against oxidative damage, and high-pigmented cells, rich in anthocyanins, would thus be able to withstand such oxidative damage. Moreover, oxidative stress would induce further anthocyanin accumulation in cells that have the anthocyanin biosynthetic pathway activated. As these cells would be able to overcome the oxidative damage, they could take advantage of the extra oxygen and increase their rate of growth. Conversely, nonpigmented cells would be less able to prevent the oxidative damage and this would result in more frequent cell death, as was observed during the in vivo imaging experiments. The latter situation could in turn affect the growth of the high-pigmented cells, which would have fewer competitors for nutrients in the culture medium.
For this to be true, the culture conditions in the 20-ml batch culture must be stressful and anthocyanins must also have an antioxidant effect in vivo.
To address the first point, it should be considered that the in vitro culture condition itself is considered by some authors to impose oxidative stress (for a review see Hagege, 1995; Cassells & Curry, 2001). High salt, water stress, mineral deficiency, overexposure to auxin and mechanical stress are situations that can cause oxidative burst in more sensitive cell lines (Cassells & Curry, 2001; Han & Yuan, 2004). In D. carota cell lines, oxidative stress was observed to occur during routine subculture of callus, as suggested by the presence of thiobarbituric acid-reactive substances (Robertson et al., 1995); in the same species, various lipid peroxidation products have been identified in dedifferentiated cell cultures, also indicating the occurrence of an oxidative stress (Bremner et al., 1997). Therefore, carrot cell lines can be considered quite sensitive to the stresses imposed by the culture conditions.
Another cause of oxidative stress in plant cell culture might be the high partial pressure of O2 compared with many plant tissues. As plants lack specialized systems for efficient oxygen distribution, many plant cells in vivo are normally exposed to very low O2 concentrations (Geigenberger, 2003).
In our system, cells are routinely grown in 50-ml volume batches, and thus are probably adapted to this situation. When cells are exposed to higher oxygen concentrations in 20 ml volumes, this could impose an additional oxidative stress similar to re-exposure to air in hypoxic plant tissues (Biemelt et al., 1998). The enhanced mechanical stress in the agitated 20-ml batch suspensions thus likely acts as a further agent contributing to cellular stress.
Anthocyanins are well-known antioxidant molecules, with an acknowledged ability to scavenge many reactive oxygen species (ROS) in vitro (Tsuda et al., 1996, 2000; Yamasaki et al., 1996; Lazzéet al., 2003). In vivo they have a role in attracting pollinators and seed dispersers, and evidence is emerging that they might also protect plant tissue against photodamage caused by ultraviolet and visible light (Klaper et al., 1996; Feild et al., 2001; Steyn et al., 2002). However, their possible role as antioxidants in vivo is less clear. Evidence exists that many plant tissues respond to different stresses by accumulation of anthocyanins (Chalker-Scott, 1999). Moreover, in vitro cultured plant cells respond to stress, such as nutrient deprivation and osmotic stress, by anthocyanin accumulation (Sato et al., 1996). Many stressing conditions, such as altered salinity (Vaidyanathan et al., 2003), herbivore or pathogen attack (de Gara et al., 2003; Mithofer et al., 2004), heavy metal exposure (Mithofer et al., 2004), and water deficiency (Farrant et al., 2003) cause oxidative stress and generation of ROS in plants. Thus, the accumulation of anthocyanins following exposure to stress could help to protect against oxidative damage. Recent evidence has suggested that anthocyanins may have a H2O2 scavenging role in planta (Gould et al., 2002). Moreover, Philpott et al. (2004) showed in situ antioxidant activity in anthocyanin-containing cells of sweet potato storage roots.
In our system, the viability of nonpigmented cells was much lower, as observed by in vivo imaging, suggesting a greater ability of the pigmented cells to adapt to the culture conditions. This variation in the viability between pigmented and nonpigmented cells, accompanied by a general decrease in cellular activities such as division, expansion and pigment accumulation, was restricted to the final stages of culture. These late stages of culture are generally characterized by sugar and mineral starvation; the latter situation is reported to be a cause of oxidative stress in plants (Malusa et al., 2002; Tewari et al., 2004). This further supports the idea that pigmented cells are better protected against oxidative damages.
In order to directly verify this hypothesis, cells from the Rosa3 culture and the K1 nonpigmented culture were treated with the glc/glc oxidase H2O2-generating system.
These experiments supported nicely the proposed hypothesis since (a) the anthocyanin-producing Rosa3 cell line is much more resistant to the induced oxidative stress than the nonpigmented K1 cell line, and (b) within the Rosa3 cell line, the nonpigmented cells are more sensitive than the pigmented ones.
The fact that the nonpigmented Rosa3 cells appeared to be much less sensitive than the nonpigmented K1 cells to the induced oxidative stress could be due to the efficient H2O2 scavenging action performed by the pigmented cells.
In conclusion, colour analysis and in vivo imaging, in combination with growth determination and biochemical analysis, allowed us to study anthocyanin production of carrot cell lines at individual cell level and to propose a model in which anthocyanin production in cell culture represents a stress response. This hypothesis, in turn, has also been verified by in vivo imaging.
This methodological approach can therefore provide a deeper knowledge of the dynamics of productivity of plant cell cultures, which is necessary in order to set up rational production strategies, since these complex systems are a source of important and high valuable secondary metabolites. Anthocyanins as well are of pharmacological and nutraceutical importance for their well-known biological properties (for a recent review see Kong et al., 2003).
However, this approach could, in principle, be extended not only to other naturally pigmented molecules but also to fluorescent molecules or molecules that can be labelled to give a coloured or fluorescent product.