The negative effect of Cd on growth parameters in V. surinamensis (Figs. 1 and 2) are in agreement with those obtained in other tree species (Zouari et al. 2016; Pereira et al. 2017; Nikolić et al. 2017).
The toxicity of Cd in V. surinamensis, more pronounced in the roots, evidenced by inhibition of its growth, especially at higher concentrations of the metal (Fig. 1A), can be explained by the direct effect of Cd and its greater accumulation in this organ of the plant (Andrade Júnior et al. 2019). In fact, in many species of plants, including the V. Surinamensis o Cd is mainly accumulated in the roots and, to a lesser extent, in the aerial part (Pereira et al. 2017; Andrade Júnior et al. 2019). This has caused root growth retardation, suberization, damage to internal and external root structures (Dai et al. 2013). The highest negative effect of Cd on the root can also be attributed to changes in cellular redox balance by the increase of reactive oxygen species (EROS) (Yan et al. 2015, Singh et al. 2016; Zouari et al. 2016) which resulted in cell death and defects in root growth and development in the zone of elongation and meristem (Abozeid et al. 2017). However, the reduction of the root system of this species was not limiting for its survival during the period of exposure to Cd.
On the other hand, the lower growth of roots, stem and leaves (Figs. 1 and 2) in V. surinamensis under Cd resulted in reduction of biomass production (Fig. 3) due to changes in photosynthesis and reduction in nutrient absorption that resulted in the low synthesis of photoassimilates. Reductions in the biomass related to the lower photosynthetic activity under Cd effect were observed by Chaves & Souza (2014). Depending on concentration, Cd may interfere with the uptake, transport and use of mineral ions by plants (Di Baccio et al. 2014). Therefore, it is suggested that Cd possibly competed with the ions by the same capture system, affecting the absorption and assimilation of the macro and micronutrients resulting in the loss of photosynthetic activity and, consequently, the reduction of dry mass (Fig. 3) in V. surinamensis. Otherwise, Cd may have affected the meristematic cells of the root and shoot, causing a decrease in the dry mass of these organs (Abdul Qados 2015). Biomass is the main indicator of energy accumulation in plants (Zang et al. 2014). Therefore, the survival of the plant under conditions of stress by Cd is dependent on a balance in the distribution of photoassimilates among its various parts. The lower shoot root ratio in V. surinamensis under the effect of Cd (Fig. 3) indicates that the growth of the root system was more strongly reduced than the aerial part. This may be explained by the higher accumulation of Cd in V. surinamensis root (Andrade Júnior et al. 2019), which may have contributed to the lower absorption and transport of macro and micronutrients (Figs. 4, 5 and 6). On the other hand, the lower ratio of root biomass to shoot can be a strategy of tolerance to the metal, reserving less energy to the roots to reduce the absorption of Cd and the greater energy investment in the leaves for the maintenance of the vital functions. Similar results were observed in other tree species exposed to Cd (Abdul Qados 2015, Silva et al. 2017, Nikolić et al. 2017).
Redution of minerals like Mg2+ (Di Baccio et al. 2014, Liu et al. 2015, Wang et al. 2016, Zouari et al. 2016), Ca2+, Fe2+, Mn2+ e Zn2+ in plants treated with Cd, probably occurs because Cd2+ competes with the membrane transporters of these minerals (He et al. 2013). In this study, the lower concentration of Mg and Fe in plants exposed to Cd (Fig. 4) indicates that the heavy metal interfered in the absorption of these nutrients in the root and in the transport to the aerial part of the plant. Probably, Cd competed with these minerals via membrane carriers (He et al. 2013), limiting the availability of Mg and Fe in the plant. In addition, Cd may have inhibited iron chelate reductase and interfered with Fe uptake (Parmar et al. 2013). Thus, in V. surinamensis exposed to Cd, the reduction of Mg and Fe may have negatively affected chlorophyll molecules, resulting in the symptoms of interverteal chlorosis (Fig. 1) and, possibly, the decrease of photosynthesis. This is because Mg and Fe, essential nutrients for chlorophyll biosynthesis (Di Baccio et al. 2014, He et al. 2013, Huang et al. 2015) may have been replaced by Cd. (Baxter et al. 2006). In the present work, it is possible to identify the presence of the chloroform molecule (Bashir et al. 2006). This may lead to an adverse effect on chlorophyll metabolism and, subsequently, the reduction of photoassimilates and the lower growth of the plant. Furthermore, Fe deficiency in plants treated with Cd negatively affects the multiprotein complex (MPCs), including photosystem II and I, LHC, Cytb6f and ATPase, (Basa et al. 2014, Bashir et al. 2015), which holds on to many electron carriers. This occurs, at least in part, because Cd replaces Iron (Fe) from its interaction with S (S) (Fe-S) sulfur in MPCs proteins (Bashir et al. 2013), possibly altering biological activity of proteins and affecting the transport of electrons to ferredoxin, resulting in the reduction of photosynthesis. On the other hand, the lower accumulation of Fe in the plants with Cd may have been a strategy of protection of the biomembranes against EROs. This is because it has been reported that the increase of Fe in plants exposed to Cd leads to the destabilization of membranes by the synthesis of lipoxygenase enzyme, since it is directly involved in the production of ROS through the reaction of Fenton and Habber-Weiss (Kumar et al. 2018). Di Baccio et al. (2014) studying tree species submitted to Cd, observed increase in Fe and Mg reduction.
Ca acts as a secondary messenger that modulates the activity of a variety of proteins (Eller and Brix 2016). Therefore, the Ca dislocation of the calmodulin protein by Cd may interfere with its ability to function correctly in signal transduction and transcriptional regulation (Dal Corso, Manara, and Furini 2013). Ca is also an essential cofactor of the inorganic catalytic core (Mn4CaOxCly) in photosystem II (PSII) and plays an important role in the stability of chlorophyll (Huang et al.2017), in the electron flux of photosystems and light dependent metabolism reactions (Hochmal et al. 2015). In addition, Ca is an essential element for the growth and development of plants (Huang et al. 2017). Therefore, it is suggested that the Cd may have substituted Ca during catalytic core formation and affected the photochemical efficiency of PSII or by competition, reducing Ca uptake by the roots, resulting in the decrease of the chlorophyll molecule and affected the photosynthetic activity of the plant, which negatively influenced the height, root growth and biomass production of V. surinamensis (Figs. 2 and 3). Reduction of Ca2+ was evidenced in other tree species exposed to Cd (Di Baccio et al. 2014).
It has been reported that Cd can damage plant DNA through the activation of restriction enzymes and / or due to the production of oxidants such as hydroxyl (OH) radical (Paunov et al. 2018). In addition, Cd can displace essential cofactors, such as Mn and Zn, and bind to functional groups (sulfhydryl, -SH) of proteins and enzymes and cause inactivation or denaturation of these organic compounds (Dal Corso et al. 2013) and may lead to various metabolic disorders (Yan et al. 2015). Thus, it is suggested that the increase of Zn (Fig. 5) in roots and leaves of V. surinamensis subjected to Cd stress would be a plant response to DNA protection by inhibition of endonucleases and the OH radical, or for protection of the -SH group, possibly to minimize oxidative damage caused by Cd to the genetic material and plant proteins. Cd stress induces changes in cellular redox balance resulting in increased ROS, such as the superoxide anion (O2 • -) (Zouari et al. 2016) that can cause oxidation of membrane lipids, proteins and nucleic acids, changes in structure (Wierke et al. 1995) and electrolyte leakage (ZOUARI et al. 2016). Thus, the increase of Zn in V. surinamensis may have played an important role in the synthesis of antioxidant enzyme, since it constitutes the cofactor of Zn-SOD associated with chloroplast (Nagajyoti et al. 2010). In a way, it could, at least in part, regulate the cellular redox potential and sustain or restore the PSII reaction center and the photosynthetic activity of the plants (Solti et al. 2016). Contrasting Zn ratio was observed in other tree species submitted to Cd (Di Baccio et al. 2014). Transport of mineral elements from soil to different tissues of plants requires different types of membrane transportes (Sasaki et al. 2016). Membrane transport proteins of the ZIP and Nramp family are involved in the uptake and translocation of Zn and Mn from the root to the aerial part of the plant at different levels (present patterns of different expressions depending on the tissues), and the Cd can inhibit these transporters (Wu et al. 2016; Akhtar et al. 2017) or Cd can compete with Zn and Mn via the cell membrane through the same uptake sites (Printz et al. 2013). In this study, the increase in Zn concentration in the root and leaves (Fig. 5) suggests that Cd did not interfere with the membrane transporters of this mineral. On the other hand, the reduction of the root Mn and the increase of this mineral in the leaves of the plants exposed to the Cd (Fig. 5), may be due to different expressions of the Mn transporters. Thus, Cd possibly may have affected the expression of the membrane transporters for Mn in the root, but no effect on the air tissue transporters of V. surinamensis. The lower concentration of Mn in the root of V. surinamensis exposed to Cd may have affected the growth and functionality of the root system. However, Mn is required in the water oxidation reaction in PSII (Schmidt et al. 2016) and its increase in Cd plant leaves (Fig. 5B) may have been a strategy to maintain stability and activity photosynthesis of PSII, although affected by Cd (Andrade Júnior et al. 2019). In addition, Mn for its role as a cofactor was possibly essential in the production of the antioxidant enzyme Mn-SOD associated with glyoxysomes (Nagajyoti et al. 2010). Mn reduction was evidenced in other tree species exposed to Cd (Printz et al. 2013).
A reduction of nitrogen (N), phosphorus (P) (He et al. 2013) and potassium (K) has been observed in plants exposed to Cd (Gomes et al. 2013). Nitrogen (N) is an essential macronutrient because it is the main constituent of many structural, genetic and metabolic compounds in plants (Kulcheski et al. 2015), such as amino acids, proteins, nucleic acids, vitamins and hormones, which play an important role in general plant growth (Singh et al. 2016). Research has shown that Cd negatively affects nitrogen metabolism due to activation or inactivation of proteins and enzymes involved in the uptake, transport and assimilation of N, resulting in reductions of N in plant tissue (Nikolić et al. 2017). Therefore, it is suggested that the reduction of N (Fig. 6) in V. surinamensis subjected to Cd doses may have caused changes in nitrogen metabolism, with a negative effect on growth (Fig. 2) and on the production of plant biomass (Fig. 4). On the other hand, N-reduction may have occurred because of its use in amino acid synthesis, such as proline, to form non-toxic Cd-proline complex in plant tissues to reduce metal phytoxicity (Chen et al.2001, Aslam et al. 2014).
P is a constituent of nucleic acids and phospholipids of the cell membrane and is indispensable for the phosphorylation reaction (Singh et al. 2016). P is important in several metabolic and physiological processes, such as the synthesis of pigments, proteins and enzymes, energetic and photosynthetic metabolism, plant growth and development (Kumar et al. 2018). Studies point to an antagonistic effect between Cd and phosphate in the solution (Cui et al. 2016). One possible explanation is that the Cd associates with the phosphate ions (H2PO4-) forming insoluble complexes Cd-H2PO4, which would limit the mobilization and bioavailability of H2PO4- (Siebers et al. 2013). Therefore, it is suggested that reduction of P in Cd-treated V. surinamensis occurred due to the sorption of Cd to P in the solution or in the cellular and subcellular compartments of the plant, which limited the availability of P (Fig. 6), as reported in other studies (Degola et al. 2014). This could possibly have interfered in the synthesis of ATP and consequently in the intracellular energy pathways (Dal Corso et al. 2013) or the formation of RubisCo, affecting the photosynthesis by limitation of the carboxylation capacity of the enzyme, resulting in the lower production of photoassimilates (Singh et al. 2016), thus causing less plant growth (Fig. 2). On the other hand, P reduction may be related to sugar synthesis through the pentoses-phosphate cycle to eliminate or keep ROS levels under control and thus repair the toxic effects of oxidants (El-Beltagi and Mohamed 2013).
The K is an essential macronutrient involved in several signaling pathways (Kulcheski et al. 2015), as in cell elongation, in osmoregulation. It activates a complex of several enzymes that aid in stomatal movement, photosynthesis, synthesis of soluble carbohydrates, protein and compounds containing soluble nitrogen (Ahmad et al. 2016, Singh et al. 2016). It is suggested that the reduction of K in V. surinamensis on Cd effect may have caused changes in the osmotic potential and interfered in water potential, in the translocation of mineral ions and amino acids or inactivating enzymes involved in photosynthesis resulting in reduction in vegetative growth (Fig. 2). The results of the present study are in agreement with those obtained by Kapoor et al. (2013).