Carbon And Nitrogen Metabolism of Young Plants of Ucuúba (Virola surinamensis) in the Presence Of Cadmium

Virola surinamensis is a forest species widely distributed in the estuaries of the Amazon. These ecosystems are susceptible to contamination by Cadmium (Cd), indicating that the plant has strategies for tolerating this metal. This study aimed to assess the nitrogen and carbon metabolism of young plants of Ucuúba (Virola surinamensis) in the presence of cadmium with the perspective of the phytoremediation of contaminated environments. The experimental design was completely randomized with ve Cd concentrations (0, 15, 30, 45, and 60 mg L −1 ) for 60 days. In general, Cd did not affect nitrate concentration in the root but had a positive effect on leaves. The reduction of nitrate reductase (NR) in plants exposed to Cd was followed by a decrease in ammonia, total soluble amino acids (TSA), and total soluble proteins (TSP). Cd promoted an increase in total soluble carbohydrates (TSC), proline, sucrose, and reducing sugars in the plants. The increase in TSC, sucrose and proline suggests a metabolic regulatory mechanism of V. surinamensis against Cd stress.


Background
Increased cadmium (Cd) concentration in the environment, caused mainly by mining residues and excessive use of phosphate fertilizers, promotes severe imbalances in terrestrial and aquatic ecosystems because of its high toxicity and persistence in the environment. Besides, this element presents high mobility in the soil for plants, being incorporated and bioaccumulated to other food chain components, rapidly affecting the growing number of organisms (Zayneb et al. 2015).
High Cd levels in the soil commonly cause many stress symptoms in plants sucs as alterations in the concentrations of starch and soluble carbohydrates in plant tissues (He et al. 2013;Elloumi et al. 2014).
It has been postulated that higher plants are more sensitive to Cd stress (Xie et al. 2014). However, a study conducted by Andrade Júnior et al. (2019) demonstrated medium and high tolerance of Virola surinamensis to Cd. Variations in Cd tolerance in plants are possibly associated with changes in nitrogen and or carbon metabolism. Moreover, they relate to differential accumulation of amino acids such as proline, and sugars, which serve as compatible osmolytes and antioxidants or are involved in other plant defense pathways against stress (Xie et al. 2014).
V. surinamensis (Ucuúba) is a forest species with economic and medicinal interest, besides being useful for the recomposition of altered areas. It is widely distributed and adapted to the lowland igapó ecosystems in the Amazon (Andrade Júnior et al. 2019). These ecosystems are frequently susceptible to heavy metal contamination, including Cd (Nascimento et al. 2006;Oliveira et al. 2015;Khan et al. 2017), indicating that the plant has strategies to tolerate such contaminated environments.
A recent study showed that, in addition to Cd tolerance, V. surinamensis had a remarkable ability to extract and accumulate metal in the root, restricting its transport to the aerial part (Andrade Júnior et al. 2019 (Fig. 1a). In the leaves, nitrate concentrations were signi cantly affected by Cd (Fig. 1b). In the roots, Cd concentrations reached 0.045 and 0.040 μmol NO 3 − g −1 DM in the control (0 mg L −1 of Cd) and at a Cd dose of 15 mg L −1 , respectively ( Fig. 1a), corresponding to a reduction of 11.11%. In the leaves, values of 0.01 and 0.02 μmol NO 3 − g −1 DM were obtained in the control plants (0 mg L −1 of Cd) and at the highest dose of Cd (60 mg L −1 of Cd), respectively (Fig. 1b), characterizing an increase of 100% in the treatment of 60 mg L −1 of Cd when compared to the control treatment.
The nitrate reductase activity (NRA) was signi cantly affected by Cd, both in roots and leaves (Fig. 1c, d).
In the roots, the lowest value (0.33 μmol NO 2 − g −1 FM h −1 ) was observed at a dose of 60 mg L −1 of Cd, representing a 56% reduction when compared to the control treatment (0.76 μmol NO 2 − g −1 FM h −1 ) (Fig.   1c). The reduction was sharper in the leaves, reaching a value of 0.02 μmol NO 2 − g −1 FM h −1 at a dose of 60 mg L −1 of Cd, corresponding to a decrease of 97.47% when compared to the control treatment (0.79 μmol NO 2 − g −1 FM h −1 ) (Fig. 1d).
Cd signi cantly affected free ammonia, both in roots and leaves (Fig. 1e,  The concentration of total soluble amino acids in roots and leaves was signi cantly affected by Cd (Fig.   2). In the roots, the concentration was 330 and 243 μmol AA g −1 DM in the control treatment (0 mg L −1 of Cd) and at a dose of 60 mg L −1 of Cd, respectively (Fig. 2a), corresponding to a reduction of 26.36% at the highest Cd dose when compared to the control treatment. In the leaves, values of 337 and 215 μmol AA g −1 DM were obtained in the control plants (0 mg L −1 of Cd) and at the highest Cd dose (60 mg L −1 of Cd), respectively (Fig. 2b), characterizing a 36.2% reduction in the 60 mg L −1 of Cd dose compared to the control treatment.
The concentrations of total soluble proteins in plants under the presence of Cd signi cantly decreased in both roots and leaves (Fig. 2c, d). The highest and lowest concentrations of proteins in roots occurred in the control treatment (0.54 mg protein g −1 DM) and at a dose of 60 mg L −1 of Cd (0.35 mg protein g −1 DM), with a 35.18% reduction at the highest Cd dose when compared to the control treatment (Fig. 2c). In the leaves, the values obtained were 0.37 mg protein g −1 DM (control treatment) and 0.15 mg protein g −1 DM (60 mg L −1 of Cd), corresponding to a decrease of 59.46% in the lowest Cd dose when compared to the control treatment (Fig. 2d).
Proline concentrations in roots and leaves of plants submitted to Cd doses increased signi cantly (

Concentration of total soluble carbohydrates, sucrose, and reducing sugars in the presence of Cd
The levels of total soluble carbohydrates in Cd-treated plants increased signi cantly in both roots and leaves ( Fig. 3a, b). The lowest and highest concentrations of carbohydrates in the roots were observed in the control treatment (0.06 mmol Glu g −1 ) and at a dose of 60 mg L −1 of Cd (0.1 mmol Glu g −1 ), with an increase of 83.3% at the highest Cd dose when compared to the control treatment ( Fig. 3a). In the leaves, the obtained values were 0.09 mmol Glu g −1 (control treatment) and 0.1 mmol Glu g −1 (15 mg L −1 of Cd), corresponding to an 11.11% increase at the lowest Cd dose when compared to the control treatment (  (Fig. 3e). The concentrations in the leaves of control plants and at a dose of 15 mg L −1 of Cd were 1.57 and 1.27 μmol carb g −1 DM, respectively, demonstrating a 19.11% reduction of reducing sugars in plants containing the lowest Cd dose when compared to the control treatment (Fig. 3f).

Discussion
NO 3 − , a crucial N source, is actively absorbed by the plasma membrane of epidermal and cortical cells of roots through nitrate carrier proteins. Still, in plants exposed to Cd, the activities of these proteins are inhibited (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, no reduction of NO 3 − occurred in the roots of most V. surinamensis (Fig. 1a); hence, the Cd probably did not affect the activity of NO 3 − carrier proteins. This result corroborates the study of Hernández et al. (2015), who showed an increase in total ATPase in the root and stem of Cucumis sativus in the presence of Cd.
In healthy plants, once absorbed by roots, NO 3 − is transported to the leaves, stored in the vacuoles, or reduced into nitrite (NO 2 − ) by NAD(P)H-dependent cytosolic NR activity (Mao et al. 2014). In this study, the increase of NO 3 − 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 NO 3 − 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 e cient energy-saving mechanism to reduce the stress and prevent the decrease of NO 3 − in the plant.
NR is the key enzyme in the process of NO 3 − assimilation (Nikolić et al. 2017) and is regulated by its presence (Van der Ent et al. 2013), degradation, activation, or inactivation. Plants exposed to Cd have a reduced NRA, leading to a decreased NO 3 − assimilation because the metal causes a lower NO 3 − 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) apparently was independent of the substrate availability (NO 3 − ) 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 also occurred in other tree species (Nikolić et al. 2017) exposed to Cd.
The ammonium ion is a central intermediate in the nitrogen metabolism in plants. It is produced during nitrate assimilation, deamination of amino acids, and photorespiration (Huang; Xiong, 2009). Since there are several biosynthesis routes for all aminoacids from ammonia (Zemanová et al. 2013), this study suggests that the decrease in ammonia levels (Fig. 1e, f) in plants submitted to Cd possibly relates to the reduction of TSA (Fig. 2a, b). Another explanation lies in the increase in the synthesis of speci c amino acids, those for 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 speci c amino acid biosynthesis, such as proline (Fig. 2e, f). Thus, the degradation of TSP could function as a self-protection mechanism combined or not with cell signaling against Cd stress. Another explanation for reduced TSP in plants exposed to Cd would be the direct effect of the metal on the NRA that affected the concentration of TSP. This assumption is supported by the signi cant positive correlation coe cient (r = 0.784; p = 0.0367) between these variables in plants under Cd, i.e., the decrease in TSP in V. surinamensis under Cd would be associated with a reduction in NRA.
The results obtained herein concerning the total soluble proteins agree with Anand et al. (2017).
The highest proline content in plants exposed to Cd occurred by the synthesis or decreased degradation or both processes (Singh et al. 2016). In this study, the increase of proline (Fig. 2a, b) in plants in the presence of Cd possibly depends on the NO 3 − concentration (Fig. 1a. b) since there was a signi cant positive correlation coe cient (r = 0.801; P = 0.0304) between these variables. On the other hand, the 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 could be a plant response to reduce the phytotoxicity of the metal (Chen et al. 2001, Aslam et al. 2014. Other forest species also showed an increase of proline induced by Cd (He et al. 2013, Yadav and Srivastava 2017. The TSC increase in V. surinamensis exposed to Cd (Fig. 3a, b) may have worked as a compatible solute, helping the plant in the osmotic adjustment against Cd stress (Singh et al. 2016). When the TSC accumulates, it may contribute to maintaining the plant's water status, favoring tissue protection and physiological processes, an essential mechanism in the tolerance of V. surinamensis to Cd, at least during the experimental period. The total soluble carbohydrate results obtained in this study corroborate with Anand et al. (2017).
Sucrose is a disaccharide consisting of glucose and fructose. Through the invertase activity, it 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 a positive effect on the activity of sucrose phosphate synthase (SPS) and a negative effect on the sucrose synthase (SuSy) caused by the metal (Fryzova et al. 2017). Besides, 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; however, heavy metals have an inhibitory effect on these enzymes (Reyes et al. 2018). The cell metabolism involving sucrose possibly reduces in plants exposed to Cd (Badr et al. 2015), accumulating this carbohydrate as an energy-saving mechanism, and improving Cd tolerance (Rahoui et al. 2015) because it chelates with sucrose. Thus, high concentrations of sucrose in V. surinamensis suggest a good metabolic regulatory state of the plant in the presence of Cd. More species exposed to Cd also contained high sucrose concentrations (Devi et al. 2007, Kapoor et al. 2016. The higher concentration of reducing sugars in plants under Cd stress (Fig. 3e, f) indicates energy savings 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). They 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. Also, 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 reducing sugar concentrations have been found in other species (Shah et al. 2017).

Conclusion
The reduction of NR, ammonia, TSA, and TSP activity evidenced the Cd effect and suggested metal toxicity, at least in part, in the nitrogen assimilation and metabolism of V. surinamensis.
The increase in TSC, sucrose, and proline suggested a metabolic regulatory mechanism in V. surinamensis exposed to Cd.

Experiment location
The experiment was conducted in a greenhouse at the Federal Rural University of Amazonia (UFRA) in Belém, State of Pará, Brazil (01°27′21″ S, 48°30′16″ W) from 15 September 2017 to 14 November 2017. According to the climate classi cation of Köppen, the climate is type Af (Tropical rainforest), with an annual average precipitation of 2921.7 mm, an average temperature of 25.9 °C, an average relative humidity of 86.8%, and a wind speed of 1.35 m s -1 .

Plant material and growth condition
Seeds of V. surinamensis were collected in the area of the Brazilian Agricultural Research Corporation (Embrapa Eastern Amazon), located in Belém, State of Pará, Brazil (01°26′44.2″ S, 48°25′03.8″ W).
Authorization is not necessary to collect seeds from this area, as it is not a Forest Reserve. At the time of collection, no botanical sample was taken for the IAN Herbarium of Embrapa Amazônia Oriental because it is a widespread species, easily identi ed by the local workers of Embrapa Amazônia Oriental on many previous occasions.
Concerning botanical veri cation, this institution's herbarium is in quarantine due to COVID-19, with no expected return. Each country has its own rules of access to its genetic resources, and in Brazil, it is more exible for Universities and Research Institutions.
These seeds were sown in 5 -L polyethylene trays containing sand and sterilized sawdust (1:1, v/v), maintained under mean air temperature (Tair) and relative air humidity (RH) of 28 o C and 90%. After emergence, the seedlings containing the rst pair of eophylls were transplanted to 10 -L polyethylene pots containing yellow latosol and poultry litter (3:1, v/v). The seedlings grown were in a greenhouse for 180 days, being irrigated daily to replace the water lost by evapotranspiration. Subsequently, the young plants were removed, and their roots washed with deionized water and transferred to 5 -L Leonard pots containing sterilized and washed sand and 800 mL of nutrient solution, replaced weekly and constituted of (

Data analysis
The experimental data were evaluated for normality and homogeneity of variances by the Shapiro-Wilk and Bartlett tests. The analyzes were performed using the SAS 9.1.3 software (SAS 2007)  The authors declare that there is no con ict of interest publishing of the paper, that the paper has not been published elsewhere, and not include any form of plagiarism. All the authors mentioned above have approved the manuscript and have agreed with the submission of the manuscript.