A common quality disorder in chicory involves the development of red discolorations on some of the leaves. A distinction is made between internal red and external red, based on the localization of the symptoms and their occurrence throughout the forcing season.

Several discoloration types can develop in the pith, which is the central core of the chicon that develops into a flowering stem when allowed to grow in the light. The most studied pith disorder is core browning, but other physiological defects such as apple pith, pink axis, and hollow pith also occur on a regular basis.

Chicons with brown edges on the external leaves are encountered rather frequently and symptoms can range from a few brown spots near the edge in the middle of the leaf (Figure 3A) to completely brown and necrotic leaf edges (Figure 3B). This color disorder is usually seen on cultivars with thin outer leaves and develops predominantly postharvest (Reerink, 1994). Leaf edge browning in chicory resembles a number of browning disorders in other crops, such as tipburn in lettuce, which are typically related to cellular disintegration as a result of calcium deficiency in young leaves that have little transpiration (Reerink, 1994; Barta and Tibbitts, 2000). However, the link between leaf edge browning and calcium has not been studied in chicory and in contrast to other crops, it occurs mainly in the oldest leaves of the chicon, which are most exposed to the environment. Alternatively, leaf edge browning could also be linked to leaf dehydration, causing mesophyll cells in the thin leaf margin to collapse, thus leading to browning of the disintegrated tissue (van Kruistum, 1992).

Past research on chicory discolorations has focused primarily on the influence of different cultivation techniques and forcing conditions on the development of color disorders. This has led to the identification of a number of influencing factors and the formulation of cultivation guidelines to minimize discolorations. In general, controlling color disorders requires a balance between yield and product quality, since conditions that promote high yield can jeopardize chicon quality. This is especially true for internal red and leaf edge browning, which are both considerably reduced by harvesting the chicons in time and by using a sufficiently low forcing temperature to prevent rapid chicon growth (Reerink, 1994; Godts et al., 2012). Likewise, the treatment of root heads with CaCl₂ before forcing has been shown to reduce core browning and hollow pith, but comes at the cost of a slightly reduced yield (van den Broek, 1994; Versluis, 1994). Although climatological conditions and soil properties can seriously influence the sensitivity to color disorders such as tipburn and pinking in lettuce (Olson et al., 1967; Hunter et al., 2017), these factors have not been reported to play an important role in the development of the most prevalent discolorations in chicory. With regard to postharvest storage, it is imperative that chicons are cooled down swiftly after harvest and stored at low temperature to minimize the development of color disorders (Godts et al., 2012). Additionally, storage under modified atmosphere conditions may further reduce the incidence of certain discoloration types, as was shown for internal red and leaf edge browning (Reerink, 1994; Vanstreels et al., 2002).

Table 1 summarizes a number of cultivation, forcing, and postharvest factors that influence sensitivity to internal red, leaf edge browning, and core browning. Some factors are specific for certain discoloration types while others, such as root ripeness, are more general. The influence of forcing and storage practices is more important than the chemical composition of the roots, especially with respect to the development of internal red (Seynnaeve et al., 2000). As a consequence, roots with an identical origin (cultivar/field/harvest date/storage conditions), may develop chicons with varying levels of discoloration when grown in different forcing facilities (van Kruistum, 1992; Coppenolle et al., 2001). Similarly, roots of variable origins will produce chicons with a different sensitivity to discolorations when forced in the same facility (van Kruistum, 1992; Coppenolle et al., 2001).

Laticifer cells are present in approximately 10% of all flowering plants and compose a tubular network filled with milky latex, which contains a broad range of specialized metabolites and defense proteins (Castelblanque et al., 2016). Although its specific function is not entirely clear, latex is thought to play a role in plant defense by creating a physical and chemical barrier against pests. Coagulated latex facilitates wound closure and pest entrapment, while secretory compounds within the latex may have an antibiotic effect (Castelblanque et al., 2020). In chicory, articulated laticifers compose an anastomosing network that is tightly associated with the phloem in the vascular bundles (Vertrees and Mahlberg, 1978). Chicory laticifers contain no phenolic compounds, but exhibit both polyphenol oxidase and peroxidase activity (Mohamed-Yasseen and Splittstoesser, 1990). The absence of phenols may explain the lack of browning in exuded latex after wounding, much in contrast to the immediate browning reaction that occurs in the polyphenol-rich latex of related dandelion species (Taraxacum spp.; Wahler et al., 2009). However, the release of latex may elicit a discoloration reaction with the phenolic compounds located in the surrounding mesophyll cells or apoplast. In lettuce, fluctuating water availability in combination with local calcium deficiency leads to overpressure in laticifer vessels, resulting in deformations of the weakened cell walls and leakage of latex into neighboring tissue, which causes the browning phenomenon tipburn (Olson et al., 1967; Tibbitts et al., 1985; Barta and Tibbitts, 2000). Similarly, in growing chicory leaves, rapid water uptake during cell elongation, is thought to cause excessive asymmetrical pressure between laticifers and the surrounding tissue. This can cause laticifer cells to rupture and bleed latex into the intercellular space, causing the elongated red spots that are typical for external red discoloration of the leaves. The rupture of laticifer cells has also been mentioned in the context of internal red and leaf edge browning. However, cells affected by internal reddening are usually situated under the upper epidermis, relatively far away from the laticifer-encased vasculature in the center or toward the abaxial side of the leaf. Moreover, it was found that the latex-exuding functionality of laticifer cells was not compromised in leaves affected by internal red discolorations (Veen, 1999). As for leaf edge browning, the association with laticifer rupture is uncertain, because this disorder occurs typically in the oldest outer leaves, in which the laticifer system has long been developed. Additionally, laticifer cells in chicory are predominantly localized on the abaxial side of the leaf, while brown edge symptoms can occur at both sides (Reerink, 1994).

Calcium ions play a critical role in the maintenance of cellular integrity by promoting cell wall rigidity and stability of the cell membranes and the middle lamellae (de Barsy and Bronchart, 1991; White and Broadley, 2003). Long-distance calcium transport to the shoot occurs via the xylem and is thereby dependent on transpiration. Subsequently, Ca²⁺ ions are further mobilized predominantly via the apoplast and enter the cell passively through Ca²⁺ channels (White and Broadley, 2003). In a range of different crops, an inadequate supply of Ca²⁺ is associated with physiological color disorders in developing tissues that have a low transpiration rate, such as young leaves, enclosed tissues, and storage organs (Den Outer, 1989; White and Broadley, 2003; Adams and Brown, 2007). For example, internal necrosis in potato, which is caused by a calcium deficiency, shows a striking similarity to pith disorders in chicory (Thornton et al., 2020). Furthermore, many leafy vegetables can develop calcium-related discolorations that resemble the symptoms of leaf edge browning in chicons, such as tipburn in lettuce (Reerink, 1994). Chicory roots, which are naturally low in calcium (±2.3 mg g⁻¹ DW), are forced under conditions of low root pressure and limited transpiration (Den Outer, 1989; Reerink, 1993). During the first half of the forcing period, translocation of calcium from the nutrient solution to the apical bud is very low and as a result, the growing chicon relies entirely on the limited redistribution of calcium from the taproot (Limami and Lamaze, 1991; Reerink, 1993). Toward the end of the forcing period, when secondary roots have developed and more water can be taken up for rapid cell elongation, calcium translocation from the nutrient solution to the chicon increases, reaching approximately 3.5–5 mg g⁻¹ DW in the harvested chicon. This is considerably lower than calcium levels in leaves of other crops, which usually range from 7 to 65 mg g⁻¹ DW (Reerink, 1994). Local calcium deficiencies have been proposed to be involved in multiple color disorders in chicory, but are particularly associated with the development of a brown pith (Den Outer, 1989; de Barsy and Bronchart, 1991). A pre-treatment of chicory roots with CaCl₂ before forcing alleviates the symptoms of brown pith, hollow pith, and point noir, but not of internal red (van den Broek, 1994; Versluis, 1994). Moreover, symptoms of pith browning are initiated during the early forcing stage characterized by low calcium uptake and mobilization, in contrast to the symptoms of internal red and leaf edge, which mainly develop during postharvest storage (Fouldrin and Limami, 1993). Calcium distribution within a chicon is comparable to other crops, with higher Ca²⁺ concentrations in older leaves and lowest Ca²⁺ levels in younger leaves (Veen, 1999). The occurrence of reddening in the medial leaves and leaf edge browning in the old leaves does not correlate with their relatively high calcium content, which makes these disorders less likely to be caused by a calcium deficiency. A dedicated analysis of the calcium content in the leaf edge, upper rib, and lower rib (where reddening usually occurs), revealed a slightly lower calcium content in the lower rib, but no relation with reddening could be deduced (Veen, 1999). However, the incidence of a temporary calcium deficiency in these tissues cannot be ruled out.

Both a surplus and a loss of water can play an important role in the development of color disorders in chicory. Excessive uptake of water for cell elongation during the final stage of forcing may lead to pressure imbalances between the continuous laticifer system and the surrounding mesophyll tissue (Reerink, 1994). Consequent laticifer rupture can then facilitate the oxidation of phenolic substrates by polyphenol oxidase, resulting in the elongated red spots that typify external red discolorations. In contrast, both internal red and leaf edge browning are more likely to result from loss of water in the affected tissue, independent of laticifers. Coppenolle et al. (2001) showed that leaves displaying the most intense reddening symptoms also have the highest water potential. After harvest, this makes them more prone to internal water redistribution toward the pith, which has a more negative water potential and keeps growing after harvest (Coppenolle et al., 2001). This process was validated by imposing an external potential pressure on harvested chicory heads by storing them with their base in either distilled water or in a sorbitol solution with a more negative water potential than the pith. This resulted, respectively, in a decrease and increase of the reddening symptoms, indicating that a strong negative water potential of the pith causes strong gradients in water content that can give rise to internal red. Similarly, postharvest storage of chicons detached from or attached to their taproot, resulted in more and less red discoloration of the leaves (Veen, 1999), confirming that water loss from the leaves leads to internal reddening. Finally, loss of water from the outer leaves due to desiccation is likely to be the driving force behind the development of leaf edge browning after harvest (van Kruistum, 1992). Differences in the transpiration rate between cultivars could help to explain the observed variation in sensitivity to leaf edge browning.

The closed conformation of a chicory head imposes some mechanical stress on the inner surface of the medial leaves. According to Veen (1999), tissue tension is maximal just below the upper epidermis and may attribute to the development of internal red discolorations by promoting the development of tears in the tissue just below the adaxial epidermis. Additionally, Gillis et al. (2001) showed that the application of a mechanical load on individual leaves induced red discoloration symptoms corresponding to the pattern of stress distribution within the leaves. Presumably, tissue tension can lead to loss of cellular integrity, resulting in unwanted discolorations in the affected tissue.

Polyphenol oxidase proteins are type-III copper enzymes that are nearly ubiquitous in higher plants. They are typically localized in the plastids and can be either soluble or membrane-bound (Martinez and Whitaker, 1995; Kampatsikas et al., 2019). Much of the research on PPOs in plants has focused on their negative impact on fresh produce quality e.g., browning reactions in apple, potato, and lettuce (Yoruk and Marshall, 2003). However, PPOs play an important role in plant defense by contributing to wound healing and immune responses against pathogens and pests (Kampatsikas et al., 2019). Additionally, some PPO isoforms have a specific function in the biosynthesis of specialized metabolites. The distribution of the PPO gene family is highly variable among plant taxa. Different isoforms show diverse substrate specificities and their expression is subject to both temporal and spatial regulation in response to environmental conditions (Tran et al., 2012; Dirks-Hofmeister et al., 2014). This flexibility allows PPO enzymes to fulfill a range of physiological functions in different tissues during different developmental stages.

Phenolic compounds are synthesized by the phenylpropanoid pathway, of which phenylalanine lyase (PAL) is the first and rate-determining enzyme (Hunter et al., 2017). Generally, the amount of PAL transcript and PAL activity is low, but expression can be induced by wounding or exposure to hormonal levels of ethylene (Tomás-Barberán and Espín, 2001; Kang and Saltveit, 2003). In chicory, phenolic compounds are highly abundant in parenchyma cells and to a lesser extent in the vicinity of the vascular tissue (Reerink, 1994). Interestingly, chicory laticifers contain no phenolic compounds as evidenced by the lack of browning in exuded latex (Mohamed-Yasseen and Splittstoesser, 1990). PPO catalyzes the first step in the oxidation of phenolic compounds to complex brown polymers or melanins, which are typically associated with color disorders in fresh produce. Depending on the type of PPO, the enzyme can catalyze the oxidation of monophenols and/or o-diphenols to o-quinones in the presence of molecular oxygen (Kampatsikas et al., 2019). These highly reactive and colored o-quinones can then lead to the formation of melanin via non-enzymatic reactions with different cellular components, such as proteins, amino acids, and other quinones (Martinez and Whitaker, 1995; Yoruk and Marshall, 2003).

Substrate specificity of PPOs toward different phenolic compounds varies considerably between species (Yoruk and Marshall, 2003). For lettuce PPO, the highest substrate specificity was observed for caffeic acid and chlorogenic acid, resulting in the formation of a pink and green quinone, respectively (Saltveit, 2018). In etiolated endive, the relative contribution of caffeic acid derivatives to the total phenolic content was shown to be increased in comparison to that of regular endive, which also developed less enzymatic discolorations than its dark-grown counterpart (Reerink, 1994). Caffeic acid and its derivatives also make up a major part of the total phenolic content in chicory, thus identifying them as potential precursors in the development of color disorders in chicons (Hanotel et al., 1995). The red color of several discoloration types of chicory may be the result of an interaction between PPO reaction products and free amino acids such as proline and tryptophan. Accordingly, the amount of free amino acids in chicory heads was found to be strongly correlated to the incidence of internal red during postharvest storage (Godts et al., 2012). Alternatively, the red pigment could result from the interaction between caffeic acid quinone and cellulose in the cell walls, which results in a stable pink complex as described in lettuce (Saltveit, 2018).

Several approaches targeting the PPO enzyme family have proven to be successful in reducing browning reactions in fruits and vegetables. Since the rate of browning depends on both PAL and PPO activity, as well as substrate availability, O₂ concentration, pH, and temperature, each of these factors can be managed in order to reduce the occurrence of color disorders (Martinez and Whitaker, 1995). As such, several postharvest approaches have proven to be successful in diminishing oxidative discoloration in fresh produce. This includes physical methods such as heat treatments, modified atmosphere packaging, storage at low temperature and irradiation, and chemical treatments such as the application of reducing agents, copper-chelating agents, natural anti-browning compounds, and acidification (Moon et al., 2020). In chicory, successful postharvest interventions have mainly aimed to tackle color disorders via the use of a sufficiently low storage temperature and modified atmosphere packaging (see also Table 1). For example, the combination of 10% CO₂ and 10% O₂ at 5°C led to a maximal reduction of internal red discoloration and additionally reduced symptoms of leaf edge browning, rot and continuation of pith growth (Vanstreels et al., 2002). One study also indicated the feasibility of reducing red discoloration of the chicon base using a heat-shock treatment of 2 min at 42°C after harvest (Jung and Kang, 2014). This reduction is thought to be achieved by the suppression of wounding-induced PAL synthesis due to a preferential synthesis of heat-shock proteins, thereby reducing accumulation of phenolic compounds and subsequent base discoloration (Kang and Saltveit, 2003). In practice, the only strategy that is consistently used to decrease discoloration incidence in chicons, is postharvest storage at low temperature, which is also essential for good shelf-life. Storage under modified atmosphere, however, is not standard practice and requires additional investments in specialized equipment.

Other than postharvest interventions, breeding strategies addressing the phenylpropanoid or PPO biosynthesis pathway could also be an effective solution (Hunter et al., 2017). There have been several successful attempts to silence PPO expression and thereby reduce discolorations in fresh produce, resulting in commercial applications such as Arctic® apple and Innate® Potato (Richael, 2021; Stowe and Dhingra, 2021). However, it is possible that the suppression of PPOs results in an increased susceptibility toward disease, due to their role in plant defense (Kampatsikas et al., 2019). Additionally, the PPO enzyme is not necessarily responsible for the full array of discolorations in a particular crop. Both in lettuce and chicory, a direct link between PPO activity, phenolic content, and the degree of discolorations could not always be observed (Jung and Kang, 2014; Saltveit, 2018). This may limit the applicability of PPO silencing in species were these enzymes play an important role in plant defense or when other specialized metabolites are at play. Alternatively, breeding efforts could target PAL or an enzyme downstream of PAL in the biosynthesis pathway of PPO substrates (Hunter et al., 2017). An improved understanding of the discoloration biochemistry and the nature of the different discoloration compounds in witloof chicory would be invaluable to identify novel targets for future breeding efforts to prevent color disorders.