Growth and Nutrient Distribution in Young Plants of Virola Surinamensis Exposed to Cadmium

The contamination of soils and water as a result of human actions has been increasingly frequent in the world, the cadmium element the as a promising contaminant of these environments. This element affects the growth and development of vegetables.The objective of the study was to evaluate the growth and concentration of macro and micronutrients in the different organs young plants of Virola surinamensis exposed to Cd. The Cd signicantly affected the growth of V. surinamensis reducing the height, stem diameter and biomass production. The Cd inuenced negatively Fe, Mg, Ca, N, P and K, especially in the root. The Zn increased in the roots and leaves, while Mn reduced in the root and increased in the leaves of the plants on exposure to Cd. The increase of Zn and Mn in the leaves may have been a strategy to maintain the stability and protection of the photosynthetic apparatus of the plant. the research concluded that cadmium affects the nutritional relationship of this vegetable, however, we could observe that the inuence of metal depends on the species being studied, the time of exposure to the metal and the amount of this metal.


Introduction
The increase in cadmium (Cd) levels and their persistence in ecosystems has aroused great concern . This is because, even at low concentrations, Cd can have toxic effects on aquatic and soil organisms, including plants and animals, and ultimately on human health (Khan et al. 2017).
Cd is a non-essential heavy metal that is readily absorbed by plant roots through essential nutrient carriers (Bashir et al. 2015), and depending on concentration may interfere with the uptake, transport and Cd, being one of the most toxic heavy metals with a high bioaccumulation capacity, has stimulated the search for solutions to remediate environments contaminated by it (Andrade Júnior et al. 2019). In this sense, phytoremediation has been proposed as one of the main promising techniques for decontamination of soils and water . Identi cation of forest species with long life cycle, large biomass production (Caires et al.2011), tolerant to and with phytoextraction capacity of Cd can serve the preservation of natural areas and the recomposition of environments contaminated by this metal.
In the Amazon, studies involving the effect of Cd on woody species are scarce (Fan et  These seeds were seeded in 5-L polyethylene trays containing sand and sterilized sawdust (1:1, v/v), and maintained at medium air temperature (T air ) and relative humidity (RH) of 28 o C and 90%. After emergence, seedlings containing the rst pair of eo los were transplanted into 10-L polyethylene pots containing yellow latosol and avian bed (3:1, v/v). The seedlings were grown in a greenhouse for 180 days, and 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 of Sarruge (1975), replaced weekly and constituted of (

Growth parameters
The height of the plant was measured, from the base of the collection to the apical bud of the plant (cm), stem diameter, measured at 4 cm in relation to the neck, using a ZAAS precision digital caliper (cm), the number of leaves was obtained by counting; for the determination of the dry matter, the plants of each treatment were taken to the Laboratory of Estudo da Biodiversidade em Plantas Superiores (EBPS), located in UFRA, where they were separated into root and aerial parts and packed in paper bags of known mass for later drying in a forced ventilation oven at 65 o C until constant mass was obtained.
Each part of the plant was weighed in an analytical balance to determine root dry mass (RDM), dry mass of the stem (DMS), dry mass of leaves (DML), dry mass of aerial parts (DMAP) and total dry mass (TDM), determinated by the sum of RDM and DMAP. With the values of RDM and DMAP the relation between RDM and DMAP (R/AP) was calcualted. After weighing, the dry matter was milled and stored in Falcon tubes and later used in biochemical analysis. Some of the dried material was taken to the Museu Paraense Emílio Goeldi (MPEG) to analyze the concentration of micro and macronutrients in the roots and leaves of the plants.

Macro and micronutrients analysis
Macro and micronutrient analysis were processed in triplicate. Magnesium (Mg), Calcium (Ca), Iron (Fe), Zinc (Zn) and Manganese (Mn) were determined according to the methodology described by Miyazawa et al. (2009). The dry matter (0.5 g) of each sample was digested in a digester tube with 8 mL of nitric acid solution (HNO 3 ) + percloric acid (HClO 4 ) (3:1). After cooling, the solution in the tube was ltered and diluted with deionized water to a nal volume of 50 mL. The Mg, Ca, Fe, Zn and Mn content were determined in this solution by atomic absorption spectrometry (Thermo Scienti c ICE 3000). Nitrogen (N), Phosphorus (P) and Potassium (K) were analyzed according to the methodology proposed by Tedesco et al. (1995). Samples of 0.2 g of dry matter were submitted to digestion with sulfuric acid (H 2 SO 4 ). The Nitrogen (N) was determined for titulation with H 2 SO 4 0,0025 M, after destillation. The P was determined by spectrophotometry and K by ame photometry.

Data analysis
The experimental data were evaluated for the normality and homogeneity of variances by Shapiro-Wilk and Bartlett tests, respectively. For the parametric variables, the means of the treatments were submitted to the PROC GLM, HSH test post hoc of Tukey utilizing the software SAS 9.1.3 (SAS, 2007). For the nonparametric variables, data were evaluated by the Kruskal-Wallis test with Bonferroni correction by the software RStudio version 1.1.383. The experimental data of all analysis were evaluated at 5% of signi cancy.

Cd effect on growth parameters
During the experiment, symptoms of metal phytotoxicity was observed as minor and necrotic roots regardless of metal concentration (Fig. 1A, B). In addition, they exhibited smaller leaves and symptoms of intercostal chlorosis (Fig. 1A, B).
The height, diameter, number of leaves and biomass of all plants exposed to Cd for 60 days were signi cantly lower than those of the control group ( Fig. 2A). The growth in height of the plants treated with Cd (60 mg Cd) was 56.8% smaller in relation to the control ( Fig. 2A). In the same treatment (60 mg

Cd effect on macro and micronutrient absorption
The concentrations of Fe and Mg in plants submitted to Cd stress were signi cantly lower than those in the control group (Fig. 4), except for Fe in the leaves at the dose of 45 mg Cd (Fig. 4B) Cd), respectively, in comparison to the control (Fig. 4).
Compared with the treatment without Cd, Zn increased signi cantly in the roots (Fig. 5D), reaching values of 42.1% in the 30 mg L − 1 Cd. In the leaves, Zn increased signi cantly at the dosages of 45 and 60 mg L − 1 Cd (Fig. 5B), reaching the value of 17.5% (60 mg L − 1 Cd) compared to the control. The Mn signi cantly reduced in the root and increased signi cantly in the leaves, except for the dosage of 30 mg L − 1 Cd (Fig.   5). In the roots Mn reduced to 43.5% (60 mg L − 1 Cd) in comparison to control. In leaves, the largest reduction in Mn was 33% (40 mg L − 1 Cd) compared to control.
Cd signi cantly affected N, P and K, both in roots and leaves (Fig. 6). In the roots, the lowest values of N,  (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 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 ux 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 e ciency 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 in uenced the height, root growth and biomass production of V. surinamensis (Figs. 2 and 3).
Reduction of Ca 2+ 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 ( (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

Conclusion
Page 10/19 Cadmium affected the uptake and translocation of Fe, Mg, Ca, N, P and K, and negatively interfered with the growth and biomass production of V. surinamensis.
The increase in Zn concentration in the root and leaves suggests that Cd did not interfere with the membrane transporters of this mineral.
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. 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.
On behalf of all authors, the corresponding author states that there is no con ict of interest.

Availability of data and material (data transparency)
Under the domain of the corresponding author