Metabolism biochemical of young plants of Ucuúba (Virola surinamensis) in the presence of cadmium

Waldemar Viana Universidade Federal Rural da Amazonia Cândido Oliveira Neto Universidade Federal Rural da Amazonia Benedito Filho Universidade Federal Rural da Amazonia Eniel Cruz Empresa Brasileira de Pesquisa Agropecuaria Cristine Amarante Museu Paraense Emilio Goeldi Vinicius Fonseca Universidade Federal Rural da Amazonia Glauco Nogueira (  glauand@yahoo.com.br ) Universidade Federal Rural da Amazonia https://orcid.org/0000-0003-3229-5694 Vitor Nascimento Universidade Federal Rural da Amazonia Diana Sousa Universidade Federal Rural da Amazonia Jessica teixeira Universidade Federal Rural da Amazonia


Background
Increased cadmium (Cd) concentration in the environment, caused especially by mining residues and excessive use of phosphate fertilizers, promotes serious imbalances in terrestrial and aquatic ecosystems because it is highly toxic and persistent in the environment, as well as present a high mobility in the soil for plants, being incorporated and bioaccumulated to other components of the food chain, rapidly affecting the growing number of organisms (Zayneb et al. 2015).
High levels of Cd in the soil commonly causes many stress symptoms in plants such as the reduction of the photosynthetic rate, which may result in alterations in the concentration of starch and soluble carbohydrates in plants tissues (He et al. 2013;Elloumi et al. 2014), and nutritional imbalance between carbon and nitrogen metabolism (Ci et al. 2009), thus reducing biomass production and total plant growth. The lower nitrate absorption (NO 3 − ) (Nasraoui-Hajaji et al. 2011;Nikolić et al. 2017), changes in nitrate reductase (NR) activity (Song et al. 2016), proline Nikolić et al. 2017), total soluble proteins (PST) and total soluble amino acids (TSA) (Rahoui et al. 2015) in plants under the effect of Cd have also been observed.
It has been postulated that higher plants are more sensitive to Cd stress (Xie et al. 2014). However, study conducted by Andrade Júnior et al. (2019) demonstrated medium and high tolerance of Virola surinamensis to Cd. Variations in Cd tolerance in plants may be associated with changes in nitrogen and or carbon metabolism. Differential Cd tolerance can be attributed 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) a forest species with economic and medicinal interest, besides being useful for recomposition of altered areas. Is a species widely distributed and adapted to the lowland and igapó ecosystems in the Amazon (Andrade Júnior et al. 2019). These ecosystems are constantly susceptible to heavy metal contamination (Khan et al. 2017), indicating that the plant has strategies to tolerate environments contaminated by heavy metals.
Recent study showed that, in addition to tolerance to Cd, V. surinamensis had a greater ability to extract and accumulate metal in the root, restricting its transport to the aerial part (Andrade Júnior et al. 2019 of Cd (Fig. 1a). In the leaves, nitrate concentrations were signi cantly affected by Cd (Fig. 1b). In the roots, Cd concentrations reached 0.045 and 0.04 µmol NO 3 − g − 1 DM in the control treatment (0 mg L − 1 of Cd) and at a dose of 15 mg L − 1 of Cd, respectively (Fig. 1a), corresponding to a reduction of 11.11% when compared to the control. 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 more accentuated 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 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, f). In the roots, values of 9.52 mmol NH 4 + kg − 1 DM (0 mg L − 1 of Cd) and 1.39 mmol of NH 4 + kg − 1 DM (60 mg L − 1 of Cd) were obtained, representing a 85.4% reduction at the highest dose of Cd when compared to the control treatment (Fig. 1e). In the leaves, Cd effect was more signi cant, promoting a reduction of 87.77% in ammonia concentration at a dose of 60 mg L − 1 of Cd (2.38 mmol of NH 4 + kg − 1 DM) when compared to the control treatment (19.47 mmol of NH 4 + kg − 1 DM) (Fig. 1f).

Effect of Cd on the concentrations of total soluble amino acids, total soluble proteins and proline concentration
The concentration of total soluble amino acids in roots and leaves was signi cantly affected by Cd

Concentration of total soluble carbohydrates, sucrose, and reducing sugars in the presence of Cd
The concentrations 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. 3b).
Sucrose concentrations in Cd-treated plants increased signi cantly in both roots and leaves (Fig. 3c, d). In the roots, the values were 1.16 mg sucrose g − 1 DM (0 mg L − 1 of Cd) and 2.11 mg sucrose g − 1 DM (60 mg L − 1 of Cd), representing an increase of 81.9% at the highest Cd dose when compared to the control treatment (Fig. 3c). The lowest and highest concentrations of sucrose in the leaves were observed in the control treatment (0.57 mg sucrose g − 1 DM) and at a dose of 60 mg L − 1 of Cd (2.38 mg sucrose g − 1 DM), with a 317.54% increase at the highest Cd dose when compared to the control treatment (Fig. 3d).
The 3. Discussion NO 3 − , 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 NO 3 − was observed in the roots of V. surinamensis (Fig. 1a), indicating that the presence of Cd probably did not affect the activity of NO 3 − 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, 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 effect of stress and not to decrease 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 the presence of NO 3 − (Van der Ent et al. 2013), its 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) did not appear to have been caused by 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. It is suggested that reduction of NRA affected NO 3 − assimilation and in uenced N metabolism, resulting in the reduction of ammonia (Fig. 1e, f), amino acids (Fig. 2a, b), and TSP (Fig. 2c, d) both in the roots and in the shoot of V. surinamensis. Reduction of nitrate reductase activity was also observed in other tree species (Nikolić et al. 2017) exposed to Cd.
The stress due to Cd activates the biochemical defense mechanisms in plants, such as sugar accumulation, which activate the NADPH-generating reactions through the pentose-phosphate cycle to eliminate or keep ROS levels under control and then repair the phytotoxic effects of oxidants (El-Beltagi et al. 2013). Thus, the increase of TSC in V. surinamensis exposed to Cd (Fig. 3a, b) may have been a protection mechanism to reduce the toxicity of this metal by ROS detoxi cation through the chelation of Cd., Moreover, a high concentration of sugars leads to a reduced mobilization of fatty acids and the accumulation of ROS is less intense (Yan et al. 2015). Therefore, an alteration in TSC can be considered as an important indicator of oxidative stress in plants. The increase in TSC 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 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 increased free proline concentration with the increasing Cd doses (Fig. 2a, b) may be related to its osmoprotective role for osmotic adjustment, which would help to diminish the water potential of tissues, avoiding dehydration.
Suggests that proline has acted on stomatal closure in order to restrict Cd absorption and translocation through transpiration, minimizing the metal damage. Proline accumulation may also have contributed to stabilize and protect subcellular structures against oxidative damage caused by free radicals , Singh et al. 2016, Solti et al. 2016. The increase of proline in plants in the presence of Cd would be due 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, Yadav and Srivastava 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 αand 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 and activation of speci c ROS elimination systems, with a subsequent reduction of oxidative damage caused by the metal and/or the sucrose would act in the osmotic adjustment for the conservation of the internal water ow of the plant, thus maintaining the water potential under su cient conditions. 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.
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). Studies conducted by Andrade Júnior et al. (2019) indicated that V. surinamensis presented medium and high tolerance to Cd. Similar results in the reducing sugar concentration have been found in other species (Shah et al. 2017).

Conclusion
In general, treatment with Cd affects nitrogen assimilation and metabolism to a greater extent in V. surinamensis leaves.
V. surinamensis presented a higher self-protection capacity in the form of bioaccumulation of total soluble carbohydrates, sucrose, and proline, important for its tolerance to the presence of Cd.

Methods
Page 8/17 To collect seeds from this area, authorization is not necessary 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 very common species and is easily identi ed.
The identi cations were carried out by the employees of Embrapa Amazônia Oriental, in relation to the veri cation of the samples, the herbarium of this institution 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 this access 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), and maintained under mean air temperature (Tair) and relative air humidity (RH) of 28 oC 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 (

Biochemical assessments
The biochemical analyses were performed at the Laboratory of EBPS of UFRA. The following variables were determined: contents of nitrate (NO 3 − ) and free ammonium (NH 4 + ) (Weatherburn 1967), activity of the enzyme nitrate reductase (RNO 3 − ) (Hageman et al. 1971); total soluble amino acids (TSA) (Peoples, 1989), total soluble proteins (TSP) (Bradford 1976); proline (Bates 1973), total soluble carbohydrates (TSC) (Dubois 1956); sucrose (Van Handel 1968); and reducing sugars (Rinner et al. 2012 The authors declare that there is no con ict of interest publishing of the paper, that the paper has been not 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.