NO3−, an important N source, is actively absorbed by the plasma membrane of epidermal and cortical cells of roots through nitrate carrier proteins, but in plants exposed to Cd, there is an inhibition of the activities of these proteins (Dai et al. 2013) because Cd damages the normal function of the proton pump (H+ ATPase) in the plasmalemma (Mehes-Smith et al. 2013, Hasanuzzaman et al. 2017). However, in general, no reduction of NO3− was observed in the roots of V. surinamensis (Fig. 1a), indicating that the presence of Cd probably did not affect the activity of NO3− carrier proteins, which is in accordance with the study performed by (Hernández et al. 2015), who showed an increase of the total ATPase in the root and stem of Cucumis sativus in the presence of Cd.
In healthy plants, once absorbed by roots, NO3− is transported to the leaves, stored in the vacuoles or reduced into nitrite (NO2−) by NAD(P)H-dependent cytosolic NR activity (Mao et al. 2014). In this study, the increase of NO3− in the leaves of V. surinamensis (Fig. 1b) suggests that Cd did not interfere with the translocation of the nitrogen compound to the shoot. The assimilation of NO3− into the cytosol of mesophyll cells may have been affected by the NRA inactivation caused by Cd. The reduction of NRA with the increasing Cd doses in the nutrient solution may be an efficient energy-saving mechanism to reduce the effect of stress and not to decrease NO3− in the plant.
NR is the key enzyme in the process of NO3− assimilation (Nikolić et al. 2017) and is regulated by the presence of NO3− (Van der Ent et al. 2013), its degradation, activation or inactivation. Plants exposed to Cd have a reduced NRA, leading to a decreased NO3− assimilation because the metal causes a lower NO3− absorption by plant roots (Nasraoui-Hajaji et al. 2011, Nikolić et al. 2017). In this study, the marked reduction of NRA with the increasing Cd concentration (Fig. 1c) did not appear to have been caused by substrate availability (NO3−) since there was no reduction of the nitrogen compound in the plant root and shoot, suggesting a direct effect of Cd on NR activity, i.e. the interaction of the metal with the thiol group (–SH) in the active site of the enzyme would result in the inactivation. Reduction of nitrate reductase activity was also observed in other tree species (Nikolić et al. 2017) exposed to Cd.
Ammonium ion is a central intermediate in the metabolism of nitrogen in plant, produced during nitrate assimilation, deamination of amino acids and photorespiration (Huang; Xiong, 2009). Considering that from ammonia, there are several biosynthesis routes for all amino acids (Zemanová et al. 2013), it can be inferred that in this study, the decrease in ammonia levels (Fig. 1e, f), in plants under Cd, it may be related to the reduction of TSA (Fig. 2a, b) or to the increase in the synthesis of specific amino acids, of protection and stress regulation, such as proline (Fig. 2e, f).
Cd stress in plants causes protein degradation and affects amino acid metabolism (Dinakar et al. 2007). The reduction in TSP (Fig. 2c, d) in V. surinamensis under Cd may be due to the activation of proteases that degrade proteins for specific amino acid biosynthesis such as proline (Fig. 2e, f). Thus, the degradation of TSP could function as an important mechanism of self-protection and / or cell signaling against Cd stress. Another explanation for the reduction of TSP in plants exposed to Cd would be the direct effect of the metal on the NRA that affected concentration of TSP. In fact, a significant positive correlation coefficient (r = 0.784; p = 0.0367) was observed between these variables in plants under Cd, ie the decrease in TSP in V. surinamensis under Cd would be associated with a reduction in NRA. The results obtained in present study in relation the total soluble proteins were evidenced by Anand et al. (2017).
The highest proline content in plants exposed to Cd occurred by de novo synthesis or decreased degradation and/or both processes (Singh et al. 2016). In this study, the increase of proline (Fig. 2a, b) in plants in the presence of Cd may be related to NO3− concentration (Fig. 1a. b), since there was a significant positive correlation coefficient (r = 0.801; P = 0.0304) between these variables, indicating that the increase in proline in plants with Cd is associated to the increase in NO3−. On the other hand, it has been reported that to degradation of proteins by proteolytic enzymes (Raldugina et al. 2016) and the accumulation of this amino acid and formation of a non-toxic Cd-proline complex in tissues would be a plant response to reduce the phytotoxicity of the metal (Chen et al. 2001, Aslam et al. 2014). The increase of proline induced by Cd was evidenced in other forest species (He et al. 2013, Wang et al. 2016, Yadav and Srivastava 2017).
The increase of TSC in V. surinamensis exposed to Cd (Fig. 3a, b) may have worked as a compatible solute, which would help the plant in the osmotic adjustment against Cd stress (Singh et al. 2016), i.e. the accumulation of TSC may have contributed to the maintenance of the water status of the plant, favoring tissue protection and physiological processes, which is an important mechanism in the tolerance of V. surinamensis to the presence of Cd, at least during the experimental period. The results obtained in present study in relation to total soluble carbohydrates were evidenced by Anand et al. (2017).
Sucrose is a disaccharide consisting of glucose and fructose and, by means of the invertase activity, plays an important metabolic role as a donor of glycosyl and fructosyl for the synthesis of polysaccharides (Sharma et al. 2006) and amino acids in plants (Todd et al. 2016). Therefore, the increase in sucrose concentration (Fig. 3c, d) in V. surinamensis exposed to Cd may be due to the inhibition of invertase activity, interfering with carbon and nitrogen metabolism, especially in proline accumulation (Fig. 2a, b). Another explanation for sucrose accumulation would be because the metal positively affects the activity of sucrose phosphate synthase (SPS) and negatively affects the sucrose synthase (SuSy) (Fryzova et al. 2017). In addition, the increase in sucrose concentration in V. surinamensis exposed to Cd may be related to the degradation of starch by the activity of the enzymes α- and β-amylase hydrolases although heavy metals have an inhibitory effect on these enzymes (Reyes et al. 2018). The higher concentration of sucrose in the plant exposed to Cd could be related to a reduction in the cell metabolism of this carbohydrate (Badr et al. 2015) as a form of energy saving since sucrose accumulation in plants submitted to Cd would be a form of tolerance to the metal (Rahoui et al. 2015), which is attributed to chelation of Cd by sucrose. Thus, high concentrations of sucrose in V. surinamensis suggest a good metabolic regulatory state of the plant in the presence of Cd. The high concentration of sucrose was also observed in other species of plants exposed to Cd (Devi et al. 2007, Kapoor et al. 2016).
The highest concentration of reducing sugars in plants under stress caused by Cd (Fig. 3e, f) indicates energy savings by plants or even the presence of Cd negatively affecting cell respiration of root and shoot. The results are consistent with those obtained by Xie et al. (2014), who suggested the increase of reducing sugars due to the lower utilization of these carbohydrates in plants exposed to Cd. The highest accumulation of reducing sugar in the root (Fig. 3e) suggests an increase in the transport of these carbohydrates from the shoot to the growing cells of the root system, indicating that Cd may not have affected the transport system of assimilates of V. surinamensis. In addition, the sugar transported to the roots because of starch degradation would be an essential energy substrate for the resumption of respiration, conferring a mechanism of tolerance of the plant against the phytotoxic effect of Cd (Rahoui et al. 2015). Similar results in the reducing sugar concentration have been found in other species (Shah et al. 2017).