Cell division and elongation processes are often inhibited in plants grown in Cu-contaminated environments (Dey et al., 2014; Tiecher et al., 2018). The inhibition of these processes was evident in both plants investigated in the current study (Figures 2 a, b, c and Figures 3 a, b, c). Beet plants were the ones mostly affected by increasing Cu concentrations; low plant development can also be associated with the significant decrease in root growth, which resulted from increased Cu content in the nutrient solution. Direct contact between plant roots and Cu ions found at toxic concentrations in the growth medium, as well as the long exposure of the root system to Cu, hinder the absorption of water and of other nutrients by plants, a fact that leads to poor plant growth and development (Ambrosini et al., 2015; Cuba-Díaz et al., 2017; Flores-Cáceres et al., 2015; Liu et al., 2014; Sánchez-Pardo et al., 2014). In addition, the excessive Cu content in the root growth medium and the increased Cu contents in the cytosol lead to higher OH- radical production via Fenton and Haber-Weiss reactions (Girotto et al., 2013; Rodrigo-Moreno et al., 2013). The high concentration of OH- radicals, in its turn, enables great opening of K+ efflux channels, as well as induces root elongation inhibition and possible cell collapse (Palm et al., 2017; Rodrigo-Moreno et al., 2013; Ryan et al., 2013).
In addition to root growth, the high Cu concentration in the growth environments and the consequent Cu absorption by plants led to significant morphological changes in the roots and tuberous root of beet plants. The increased diameter and brownish color of the roots (Figure 4) was likely associated with excess of Cu in the root tissues, a fact that led to changes in distal regions of the root apex, as well as increased diameter in root areas presenting cortical and vascular cylinders (Ambrosini et al., 2015; Michaud et al., 2008). The larger root diameter of beet plants grown under high Cu concentration may also result from root growth inhibition caused by disturbances in endoderm differentiation and by premature cortical tissue lignification (Ambrosini et al., 2015; Kováč et al., 2018).
Unlike beet plants, cabbage root growth was not hindered by increased Cu concentrations in the culture medium. This response may be associated with the greater regenerative power of the cabbage root system and, mainly, with the great ability of this species to accumulate and tolerate high Cu levels in the root (Figures 3 c, e and f) (Radulescu et al., 2013).
Beet plants grown in the nutrient solution subjected to the highest Cu concentration presented the following trend of Cu concentrations in their tissues: roots > leaves > tuberous root > petiole, whereas cabbage plants followed the following trend: roots > stem > leaves > head. On the other hand, Cu accumulation in beet organs presented the following trend: tuberous root > roots > leaves. The highest Cu accumulation in tuberous roots was directly associated with higher biomass production (Figure 2b), whereas the highest Cu accumulation in the root may have resulted from higher Cu concentrations in it. Cabbage plant roots accumulated 60% of the Cu absorbed by the plant. Although Cu concentrations in cabbage head leaves increased as Cu in the nutrient solution increased, the amount of Cu accumulated in this organ did not significantly change (Figure 3 f). This outcome can be attributed to lower head growth, which was assessed through variable HDM.
The increased Cu retention in the root system of both plant species evidenced high Cu affinity for carboxylic groups found in cell wall. Some Cu may have been retained in the root apoplast, and it reduced Cu concentration in the symplast (Ambrosini et al., 2018, 2015; Comin et al., 2018). The increased Cu concentration in plant roots also results from intracellular production of organic acids such as citrate (Keller et al., 2015; Murphy et al., 1999), as well as of phytochelatins acting in cytosol. During the metal ion chelation process, chelated metals are sequestered and compartmentalized in the vacuole (Mourato et al., 2015) to avoid detrimental effects on cell metabolism (Jan and Parray, 2016; Tiecher et al., 2018). The preferential Cu accumulation in the root system is essential to prevent excessive Cu contents in the shoot, mainly in leaves.
Although the highest Cu retention was found in the root system of both vegetables, shoot organs also recorded increased Cu levels. Beet and cabbage leaves reached concentrations higher than 20 mg Cu kg-1 dry mass (DM) when they were exposed to Cu concentrations in the nutrient solution higher than 0.27 mg L-1 and 0.36 mg L-1, respectively. Cu concentrations between 15 mg kg-1 and 20 mg kg-1 of DM in leaves are referred to as limiting to the growth of Cu-sensitive plants (Kabata-Pendias, 2011). This aspect can lead to several physiological disturbances in plants, since high Cu concentrations in leaf tissues can affect the transport function of the membrane and of ion channels because it changes membrane properties (Janicka-Russak et al., 2008). Changes in the nutritional balance of plants exposed to high concentrations of heavy metals result from increased non-specific membrane permeability (Cambrollé et al., 2013). This aspect contributes to increased Cu absorption and accumulation in plant tissues (Tiecher et al., 2018), since excessive Cu can compete with other cations of the same valence, such as Fe and Mg, in root absorption, as well as in other assimilation sites (Xu et al., 2015).
The increased Cu concentration in shoot organs resulted in higher peroxidase (POD) activity in the leaves of beet plants (Table 1), and in higher superoxide dismutase (SOD) activity in the leaves of cabbage plants (Table 2). High Cu contents in leaf tissues help catalyzing Haber-weiss reactions, which increase the production of reactive oxygen species (ROS) (Karimi et al., 2012; Miotto et al., 2014; Tiecher et al., 2018). Some defense genes may have induced POD and SOD expression, and increased their activity in beet and cabbage leaves, respectively, in response to excessive ROS production. Such response may differ between plant species, as well as between tissues in the same plant. (İşeri et al., 2011; Passardi et al., 2005). This outcome indicates the potential of these enzymes to mitigate oxidative damages, since SOD act in superoxide (O2•-) conversion into H2O2 (Gill and Tuteja, 2010; You and Chan, 2015), which is often correlated to increased plant tolerance (Sharma et al., 2012). POD can directly convert H2O2 into H2O and O2; thus, it plays a key role in enabling responses to abiotic and biotic stress (Karuppanapandian et al., 2011; Yu et al., 2017), as well as in suppressing cell damages (Wu et al., 2014).
Normal TBARS and H2O2 levels were observed in the leaves of plants throughout the experimental period. This outcome has shown that the antioxidant system was efficient in controlling the antioxidant imbalance trend, which was explained by increased POD and SOD activity in beet and cabbage plants, respectively. However, maintaining homeostasis is costly to stressed plants, such distress can be expressed by the decreased dry matter yield in these plants, which indicates that the plant may have demanded energy to maintain the antioxidant system.
The tolerance of the investigated vegetables to increased Cu concentrations in the culture medium was evaluated based on CTC, TC25 and TC50. Results indicated that cabbage plants are more tolerant than beet plants; cabbage CTC and TC25 values were at least 50% higher than values recorded for beet plants. In addition, TC50 was not reached in tuberous roots of beet plants or in any cabbage plant tissue; it may have happened because the critical concentrations exceeded the Cu concentrations tested in the current study. Higher cabbage tolerance may be associated with mechanisms triggered to mitigate damages resulting from excessive Cu contents in the plant culture medium. Among these mechanisms one finds Cu complexation/chelation and subsequent compartmentalization in the cell vacuole (Mourato et al. 2015), as well as the action of antioxidant enzymes (Karimi et al., 2012; Miotto et al., 2014).
It is possible estimating the daily Cu intake based on the consumption of such vegetable if one takes into consideration the Cu accumulation trends presented by cabbage plants, the decreased biomass production of tuberous roots and cabbage heads, as well as the Cu contents found in these tissues. Therefore, based on a daily per capita intake of 50 grams of each vegetable in separate, and by comparing the recommended daily Cu intake (RDI) for adult individuals - which is 0.9 mg in Brazil (Brasil, 2005) and in the USA (FDA 2001) -, it is possible stating that tuberous roots of beet plants grown in nutrient solution subjected to Cu concentration of 1.5 mg L-1 account for 23% of the RDI, whereas the intake of cabbage heads of plants grown in nutrient solution subjected to Cu concentration of 2.52 mg L-1 represent approximately 16.5% of the RDI.