Nova Biotechnol Chim (2017) 16(2): 99-104 DOI: 10.1515/nbec-2017-0014  Corresponding author: bardacova.monika@gmail.com  Nova Biotechnologica et Chimica Variable dynamics of cadmium uptake and allocation in four soybean cultivars Monika Bardáčová1,, Yevheniia Konotop2, Zuzana Gregorová3, Miroslav Horník1, Jana Moravčíková3, Ján Kraic4 and Ildikó Matušíková1 1 Department of Ecochemistry and Radioecology, Faculty of Natural Sciences, University of Ss. Cyril and Methodius in Trnava, Nám. J. Herdu 2, Trnava, SK-917 01, Slovak Republic 2 Department of Plant Biology, Educational and Scientific Center “Institute of Biology and Medicine”, Taras Shevchenko National University of Kyiv, Volodymyrska 64, Kyiv, 016 01, Ukraine 3 Institute of Plant Genetics and Biotechnology, Centre of Biology and Biodiversity Slovak Academy of Sciences, Akademická 2, P.O. Box 39A, Nitra, SK-950 07, Slovak Republic 4 Department of Biotechnology, Faculty of Natural Sciences, University of Ss. Cyril and Methodius in Trnava, Nám. J. Herdu 2, Trnava, SK-917 01, Slovak Republic Article info Article history: Received: 12th October 2017 Accepted: 16th December 2017 Keywords: Cadmium Distribution in tissue Glycine max Metal intake Radiotracers Abstract Cadmium is a serious environmental pollutant and its uptake by plant represents a serious health risk. Uptake, accumulation as well as sensitivity of soybean plants to metals have been shown to vary with genotype, while the dynamics of this uptake has rarely been studied. Here we studied the uptake and accumulation of Cd2+ ions in different parts of soybean plants of four cultivars Moravians, Gallec, Kent and Cardiff. The plants at early developmental stage were immersed in Hoagland nutrient solution in the presence or absence of 50 mg.L-1 and the isotope of 109Cd2+ to monitor its accumulation continuously at 24 h intervals for 10 days. Our results showed that the uptake rate varied among the cultivars, being the highest in roots of the cv. Moravians and the lowest in the cv. Gallec. We also observed a non-even distribution of radioactivity within the entire plants of individual cultivars. The most of Cd2+ isotope was translocated into primary leaves and leaves in the cvs. Kent and Moravians; on the contrary, relatively less in the cvs. Cardiff and Gallec. The results were fitted with genetic potential, growth as well as defense parameters such as proline accumulation. Combining uptake dynamics and biochemical data are indicative for different tolerance strategies of soybeans.  University of SS. Cyril and Methodius in Trnav Introduction Contamination of soils represents a serious concern of sustainable environment but also food production. Heavy metals, released to the environment from natural sources (e.g. volcanic activity, weathering of rocks) but in great manner also due to various anthropogenic activities (mining, industry etc.), endanger plants but also animals including humans. Therefore, metal contamination of farmland has widely been discussed due to its potential risk for food safety (Zhao et al. 2017). Cadmium toxicity represents one of the most severely discussed safety issue in many European countries. For example in Slovakia, an average Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 08:00 UTC Nova Biotechnol Chim (2017) 16(2): 99-104 100 concentration of 1.24 mg Cd per kg of soil was reported (Granec and Šurina 1999; Six and Smolders 2014) comparing to 0.4 mg Cd.kg-1 worldwide. The exposed organisms are primarily plants, which have to face metal-induced membrane disruption and electrolyte leaking, disruption of water balance, oxidative stress, inhibition of enzymes and others, leading to impaired plant processes and finally reduced yields. The workable strategies to address Cd toxicity in plants include selection of genotypes that either demonstrate low uptake or are hyperaccumulators (Jha and Bohra 2016). For several crop species including rice, wheat and soybean have already been identified genetic markers that allow for selection of genotypes with low genetic potential to accumulate cadmium (Jegadeesan et al. 2010). In addition to many transporters and channels on the plasma membrane (Van Kerkhove et al. 2010), the metal uptake largely depends on the composition and properties of cell walls since they affect metal binding (immobilizing) and filtering capacity, restricting metal entrance to the cells (Parrotta et al. 2015). The metal uptake and its distribution in plants has widely been studied, however, reports on dynamics of these processes are rather rare. This work was devoted to studying the dynamics of Cd uptake in four soybean cultivars, and to reveal its distribution within the plants. Our findings show that the uptake of Cd by soybean roots is variable among the cultivars and might significantly contribute to the ability of soybeans to cope metal toxicity. Experimental Plant material and experimental conditions Soybean (Glycine max L.) seeds of four cultivars Moravians, Cardiff, Gallec and Kent were obtained from Matex, s.r.o (Veľké Kapušany, Slovakia). Seeds were sterilized with 0.5 % (w/v) sodium hypochlorite for 5 minutes and germinated on wet filter paper in Petri dishes. After 2 days, uniforomly germinated seeds were placed into 5 mL vial tubes with ¼ Hoagland solution with presence or absence of 50 mg.mL-1 Cd2+ in the form of CdCl2. The seedlings were incubated in dark at 23 °C for 48 hours. At daily time intervals the roots were subjected to analyses. At the end of experiment (10 days) the tolerance indexes (TI) were determined for each cultivar as ratio of dry weight of control to metal-exposed tissue x 100. All determinations were performed in triplicate. After verification of normal distribution and variance homogeneity the data were analysed by t-test. Determining the genetic potential for Cd accumulation The simple sequence repeat (SSR) genetic markers amplifying the Cda1 locus for low Cd accumulation capacity (Jegadeesan et al. 2010) were used. DNA was isolated according to Békésiová et al. (1999). The PCR conditions were described in Socha et al. (2015). Reproducible presence/absence of amplicons in at least two of the 3 marker primers SatK147, SatK 149 and SatK 150 (Jegadeesan et al. 2010) was taken for evaluation of accumulation potential. Content of proline Content of proline in plant tissues upon homogenization with liquid nitrogen was determined spectrophotometrically as described by Bates et al. (1973). Radiometric analysis Determinations of radioisotope 109Cd in liquid samples of culture medium (expressed as Bq/mL) or in solid samples of washed plant biomass (expressed as Bq/g tissue) was implemented by scintillation gammaspectrometry with well type NaI(Tl) crystal, using a scintillation gammaspectrometer, type 76BP76/3 (Scionix, The Netherland). Calibration of the instrument and calculation of radioactivity were realized using a library of analyzed radionuclides and the program ScintiVision-32 (Ortec, USA). Results The genetic potential for accumulation of cadmium was determined for the tested varieties. The data showed that the Cda1 locus was present in only the Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 08:00 UTC Nova Biotechnol Chim (2017) 16(2): 99-104 101 variety Gallec, which therefore we assigned as of low Cd accumulation potential (Fig. 1). The tolerance indexes for growth in presence of Cd showed highest values for the cultivars Kent and Gallec, while the variety Moravians was the most sensitive one (Table 1). To estimate the stressed nature of tissues we measured the levels of proline as typical multifunctional metabolite accumulating Fig. 1. PCR-detection of the locus Cda1 for low Cd accumulation capacity. Three primers including SatK150 (A) were used, and reproducible presence of corresponding amplicons for at least two of them conditioned the assignment of a genotype as a low metal-accumulating type (B). Table 1. Relative values of individual parameters in soybeans exposed to 5 mg/L of cadmium. Variety Genetic potential to accumulate Cd Tolerance index (%) Proline SD Moravians high 62 1.1780* 0.5434 Cardiff high 75 1.2243 1.5104 Gallec low 78 1.7488** 4.5375 Kent high 80 1.3125*** 5.2231 Statistically significant differences are marked * at P ≤ 0.05 and *** P ≤ at 0.001. in plants under metal stress. Significant elevation was observed in each cultivar, except for the variety Cardiff (Table 1). With radiological analyses, we studied the uptake, accumulation and allocation of cadmium in different parts of soybeans. The plants were immersed in Hoagland nutrient solution in the presence or in the absence of the defined amount of the isotope 109Cd2+. First, its depletion from solution by roots was measured continuously at 24 h intervals for 10 days. Our results showed that the uptake rate greatly differs among cultivars. Fastest and most intense cadmium uptake (removal) from the solution was typical for the cv. Cardiff already after 1 day and Moravians after 2 days (Fig. 2A), in contrast to the cvs. Gallec and Kent. In the latter two varieties the uptake was clearly delayed and ~10 times lower after 2 days than in the cv. Moravians. The difference in the uptake rates from environment (solution) among the varieties Moravians/Cardiff and Gallec/Kent decreased with time, nevertheless still remained more than ~ 2 fold at the end of the experiment (Fig. 2A). The two former cultivars removed up to 85% of cadmium from the solution, while the latter ones only 50 – 60% (Fig. 2). Part of cadmium taken up by plants from the solution accumulated in the plant tissue. Due to different physiology we again detected differences among tested cultivars. Highest radioactivity representing the amount of cadmium accumulated in the tissue was detected in the cv. Cardiff (Fig. 2B). The Cd contents in the other three cultivars (including the cv. Moravians) were comparable to each other, but obviously lower comparing to the cv. Cardiff (Fig. 2B). The differences in metal deposition in tissue among the cultivars were not as pronounced as in case of the uptake rate from solution; while up to only 5 fold difference was observable after 2 days, at the end of experiment the amount of cadmium deposited in tissues of all Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 08:00 UTC Nova Biotechnol Chim (2017) 16(2): 99-104 102 cultivars was in a range of 120 – 170 µg/g of tissue (Fig. 2B). The total amount of Cd accumulated in the plant tissue during 10 days was determined. Highest total amount of cadmium accumulated in the cv. Cardiff, in contrast the lowest amount was detected in the cv. Moravians (Fig. 3A). Fig. 2. The dynamics of uptake (depletion) of 109Cd2+ from the growth solution by the root system of four soybean cultivars (Cardiff, Gallec, Moravians, Kent) after 9 days of culturing in the liquid Hoagland medium. Intake of Cd2+ by the tissue is expressed in % of the initial amount of Cd2+ solution (A) and in mg/g (dry weight) (B). Data represent the average values obtained from 3 independent experiments. The metal was distributed within the soybean plants, while the allocation pattern depended on cultivar. The cv. Moravians retained most of metal in roots, in contrast the cv. Kent translocated a considerable amount of Cd2+ into aerial parts – mainly to the stem and to primary leaves (Fig. 3B). Discussion Sensitivity/tolerance of plants (including soybean) to cadmium results partly from genetic potential. Therefore, uptake of the metal from environment and the ability of plants to alleviate toxicity vary among genotypes/cultivars. Chromosome loci have been identified (Benitez et al. 2010; Jegadeesan et al. 2010) that can explain to some extent the variability in tolerance to cadmium. In our study, the locus for low cadmium accumulation potential Cda1 was present in the cultivar Gallec (Fig. 1), and confirmed (radio)analytically (Fig. 3A). In this cultivar, the metal uptake is probably governed mainly by the transporters (and other genes) on the chromosome 9. However, comparable (or even lower) amounts of metal did accumulate in the other tested cultivars, in which the Cda1 locus was absent. This is in agreement with the fact that the given locus explains ~60% of observed variability for soybean tolerance to cadmium (Jegadeesan et al. 2009; Socha et al. 2015), and suggests for these cultivars another mechanisms regulating the metal uptake. The dynamics of cadmium entrance to the root cells (and their compartments) can vary, depending on several factors including pH of soils and metal speciation (Rodriguez-Serrano et al. 2009), or metal concentration in the growth media (Zhao et al. 2017). At given dose we observed a gradually increasing uptake of Cd with time during the 10 days of experiment. The uptake of metal from solution was very rapid in case of the cvs. Cardiff and Moravians (Fig. 2A), but the metal amount measured in the tissue was correspondingly high only for cv. Cardiff (Fig. 2B). Interestingly, this cultivar appears as relatively tolerant to Cd, while under similar conditions we observed lack of induction of defense components such PR protein synthesis (Bardáčová et al. 2016) or proline accumulation (this study). In addition, some part of cadmium was transported to the areal parts of plants (Fig. 3B). We propose that the cv. Cardiff might have an efficient system for detoxification and/or intracellular sequestration of cadmium ions, e.g. high level of phytochelatins, glutathion or organic acids (Schützendübel and Polle 2002). Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 08:00 UTC Nova Biotechnol Chim (2017) 16(2): 99-104 103 Fig. 3. Cadmium allocation in four soybean cultivars (Cardiff, Gallec, Moravians, Kent). Total amounts of Cd2+ deposited in soybean plants were measured (A) and the relative abundances within different plant parts was calculated (B). The data represent average values of three independent experiments from plants after 10 days of culturing in liquid medium with 50 mg/L Cd2+ and spiked with 109CdCl2 (141.7 kBq/L). Among the tested soybeans, the cv. Moravians took up Cd2+ from solution as rapidly as Cardiff, however, accumulated Cd in the tissue like the cvs. Gallec and Kent (Fig. 2B).Moreover, after 9 days we quantified in this cultivar the lowest amounts of Cd of all tested genotypes. Noteworthy that Gallec is genetically (Socha et al. 2015; this study) and also practically (this study) a low-Cd accumulator. Therefore, we conclude that part of Cd ions likely binds to the cell walls on the surface of roots (Cataldo et al. 1983; Piršelová et al. 2012) but never enters the root. This fits with relatively high tolerance index of this cultivar, too. The allocation pattern of Cd in soybean plantlets points to even higher complexity of metal transport in plats than expected. Furthermore, metal distribution and allocation within plants reflect to different strategies applied by soybeans to withstand negative impacts, as suggested previously (Arao et al. 2003). The cv. Gallec exerted low genetic predisposition for cadmium accumulation, and the both uptake dynamics and real accumulation rate supported the molecular prediction. In contrast, the cv. Moravians with relatively slow but intensive uptake and translocating the metal to shoot revealed the poorest strategy resulting in high sensitivity to cadmium. Dynamics of uptake as well as deposition rate and allocation of metals within a plant might be indicative to defense efficiency and strategy of a plant genotype to cope metal toxicity. Conclusions Sensitivity of soybean plants to cadmium can partly be explained by genetic determinants. However, uptake of the metal from environment varies among cultivars. Furthermore, metal distribution and allocation within plants reflect different strategies applied by (soybean) plants to withstand negative impacts. The cv. Gallec exerted low genetic predisposition for cadmium accumulation and the both uptake dynamics and accumulation rate supported the molecular prediction. In contrast, the cv. Moravians with relatively slow but intensive uptake and translocating the metal to shoot revealed the poorest strategy resulting in high sensitivity to cadmium. Dynamics of uptake as well as deposition rate and allocation of metals within a plant might be indicative to defense efficiency of a plant genotype to cope metal toxicity. Acknowledgement This work was supported by the bilateral project SK-BG- 2013-0007 and by the project APVV-15-0051. We gratefully acknowledge the financial support for the stay of YG at UCM Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 08:00 UTC Nova Biotechnol Chim (2017) 16(2): 99-104 104 in Trnava provided by Slovak Academic Information Agency SAIA. References Arao T, Ae N, Sugiyama M, Takahashi M. (2003). 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