ORIGINAL ARTICLE

Plasma Membrane Alterations in Phaseolus vulgaris L. Variety CC-25-9-N Induced by Metals

Dr.C. Liane Portuondo Farías^{I  *} 

Dr.C. Dariellys Martínez Balmori^I 

Dr.C. Fernando Guridi Izquierdo^I 

Dr.C. Alejandro Falcón Rodríguez^II 

Dr.C. Andrés Calderín García^III 

Dr.C João Paulo Machado Torres^IV 

^IUniversidad Agraria de La Habana, Departamento de Química, San José de las Lajas, Mayabeque, Cuba.

^IIInstituto Nacional de Ciencias Agrícolas, San José de las Lajas, Mayabeque, Cuba.

^IIIUniversidade Federal Rural do Rio de Janeiro, Laboratório de Solos, Rio de Janeiro, Brasil.

^IVUniversidade Federal do Rio de Janeiro, Laboratório de Radioisótopos, Rio de Janeiro, Brasil.

ABSTRACT

It is in the early stages of the vegetative phase, when the plant manifests its greatest susceptibility to sudden changes in environmental conditions and unleashes adaptive pathways that include changes in the structure of cell membranes, as they are the first to respond to damage. The bean (Phaseolus vulgaris L.) is a crop of agricultural interest that can accumulate heavy metals and therefore, the possibility of developing mechanisms of tolerance to this type of stress. For these reasons, the work aimed to determine the existence of changes in the plasma membrane and in the antioxidant activity of leaves in bean plants germinated in presence of heavy metals. Seeds of the variety CC-25-9-N were germinated under controlled conditions with six specific concentrations of Pb²⁺, Ni²⁺ and Cu²⁺ cations. Fifteen days after seed´s germination, the tolerance limit, the lethal dose and changes in the permeability of the leaf membranes were determined through electrical conductivity, pH and the activity of total peroxidases and guaiacol peroxidase. The variability in the membrane permeability was checked from differences in the values of the electrical conductivity and the pH in each of the treatments, which depended on the metal and the concentration under study, as well as on the activity of the enzymes analyzed. These results suggest a possible specificity in the plant - metal interaction that implicate different response mechanisms.

Keywords: beans; toxic metals; oxidative stress; toxicity; peroxidases

INTRODUCTION

Stress conditions are one of the main factors that influence the normal growth and development of plants. Various pollution processes that include anthropogenic action or factors of the natural environment such as changes in salinity, extreme temperatures, drought or high intensity of light can unleash them. Plants respond to these mismatches through natural defense systems that allow them to counteract external conditions, as well as the incidence of biotic and abiotic factors (^{Shivaraj et al., 2018}).

Soil contamination by metals is one of the most serious environmental problems caused by man. Some metals such as copper (Cu), zinc (Zn), nickel (Ni) and manganese (Mn) are essential micronutrients for plants, while others such as cadmium (Cd) and lead (Pb) do not have a known function. The excess of these metals implies damage to the plant and an additional danger if they accumulate and integrate into the food chain. Minimizing the entry of these pollutants into the food chain is an imperative task for sustainable development (^{Pirzadah et al., 2015}).

Although plants control the absorption or rejection of some chemical elements by specific physiological reactions, they are passive receptors of trace elements absorbed by the roots or those that arrive by precipitation. The studies carried out on this topic show results that involve adaptation mechanisms such as: (i) joining metals to the cell wall of the roots and (ii) activation of a reduced transport through the inner membrane of the cells. Others are (iii) active entry or exit into the cellular interior; (iv) compartmentalization within the vacuoles and (v) chelation through the union of the metal with organic, inorganic complexes and specific proteins such as phytokelatins or metallothioneins (^{Hasan et al., 2017}).

Several plant species have been reported with possibilities for the accumulation of toxic metals in their tissues, including some of agricultural interest like beans. This crop shows an accumulation of toxic cations mainly in the roots, not detected in the agricultural fruit (^{Portuondo, 2011}). It is known that plants in the early stages of the vegetative phase, show greater sensitivity to changes in homeostasis, or have the greatest expression of the possible adaptive mechanisms. Therefore, the objective of this work was to determine the existence of changes in the plasma membrane and in the antioxidant activity in the leaves from germinated bean plants, in presence of heavy metal cations Pb²⁺, Ni²⁺ and Cu²⁺.

METHODS

Materials and Experimental Conditions

The experiments were carried out in the vegetative phase of Phaseolus vulgaris L. plants, under laboratory conditions, in the Chemistry Department at the Agricultural University of Havana. The plants were obtained from seeds of the CC-25-9-N variety, certified by the National Institute of Agricultural Sciences (INCA), of which no report was found on their ability to accumulate metals.

Establishment of Tolerance Limit to Pb²⁺, Ni²⁺ and Cu²⁺ cations

Twenty seeds were germinated in plastic trays of 9 × 13 cm with the corresponding solutions of Pb(NO₃)₂, NiSO₄.6H₂O and CuSO₄.5H₂O (high quality, Sigma-Aldrich) at six different concentrations shown in Table 1 and taking into account permissible values previously reported (^{Kabata, 2001}). A control treatment (water) was also used for 19 treatments, with five replicas each.

TABLE 1 Concentrations of the Pb²⁺, Ni²⁺ and Cu²⁺ metal solutions 

ppm
Pb ^2+	10	25	50	75	100	125
Ni ^2+	1	5	10	15	20	25
Cu ^2+	5	10	20	30	40	50

Fifteen days after seed germination (DAG), the mean lethal dose (MLD) and phytotoxicity index (PI) evaluations were performed. The MLD was considered as the one that destroyed 50 % of the individuals in the population and the PI as that corresponding to the number of affected plants (^{Rivera et al., 2005}).

Evaluation of Changes in Membrane Permeability

It was performed through measurements of pH and electrical conductivity. Ten discs of 0.5 cm in diameter were taken in triplicate from the true leaves of five plants of each treatment, which were introduced into containers with 30 mL of deionized water. The system was kept under constant stirring (magnetic stirrer) for 30 min, after this time reading of both variables were made every 5 min at a constant temperature of 25 °C (^{Schulze et al., 2005}). The pH of the solution was measured in a pH meter (Model PHSJ-3F, 0.01pH ± 1bit) and the electrical conductivity in a conductometer (Model DDSJ-308, ± 0.5% (FS) ± 1digit).

Influence of Metal Cations on Antioxidant Systems

Bean seeds to germinate were placed in plastic trays as it was described above, with two concentrations of the metal cations (corresponding to the MLD and the Limit of Tolerance) and a control treatment (water), for seven treatments with five replicas each. The activity of peroxidases enzymes (POX) (EC: 1.11.1.7) was evaluated, which was determined at 15 DAG from a mass of 0.5 g of leaves. A reaction mixture consisting of Na₂HPO₄-NaH₂PO₄ 0.05 mol L^-1 pH 5.5, guaiacol 0.05 mol L^-1, H₂O₂ (2 % v: v) and 1 mL of an enzyme extract prepared from the addition of the same buffer at pH 5.8, 5 % PVP, filtered and centrifuged at 10,000 rpm. The absorbance reading was performed at 470 nm for 4 min at 20 sec intervals on a UV-visible spectrophotometer (Rayleigh-1601). The results were expressed in Δ₄₇₀ min^-1 g^-1 protein (^{Bronikowski et al., 2018}).For guaiacol peroxidase (GPX) (EC: 1.11.1.7) the same mass of plant material was used and the same reaction mixture was prepared with the difference that the buffer used was at a value of pH 6.1 and pH 7.0 for the enzymatic extract. The absorbance reading was performed at 420 nm and the results were expressed in ∆₄₂₀ min^-1 g^-1 protein (^{Wilkesman et al., 2014}).

Data were processed using a simple classification ANOVA with the Statgraphic statistical package v. 5.1 plus, and the means comparison test was performed by Tukey p<0.05.

RESULTS AND DISCUSION

Establishment of Tolerance Limits for Pb²⁺, Ni²⁺ and Cu²⁺ Cations

The mean lethal dose (MLD) values for the Pb²⁺, Ni²⁺ and Cu²⁺ cations, where germination of 50 % of the seeds was not reached, were 125, 10 and 30 ppm, respectively. Another indicator to consider is the phytotoxicity index (PI), which represents the plant's response to exposure to metals. The higher PI value represents a greater phytotoxic or stress effect of the metal, while the smaller means the opposite. PI values in bean seedlings exposed to cations of Pb²⁺, Ni²⁺ and Cu²⁺ are shown in Figure 1.

FIGURE 1 Phytotoxicity index (PI) of bean seedlings (Phaseolus vulgaris L.) variety CC-25-9-N with 15 DAG in solutions at different concentrations of toxic metals Pb²⁺, Ni²⁺ and Cu²⁺. 

For the treatment with Pb²⁺, the PI responds with higher values to the highest concentration of 125 ppm. However, in the case of Ni²⁺ and Cu²⁺ at concentrations below the maximum, a notable PI is already observed. This new and useful result indicates that bean plants, in their early stages of the vegetative phase, tolerate higher concentrations of Pb²⁺ than other metals such as Ni²⁺ and Cu²⁺. This is probably because Pb²⁺ does not present any recognized biological function in plants, so that when introduced into the cell, detoxification mechanisms are activated and rapid accumulation in vacuoles occurs (^{Shah et al., 2018}).

In the cases of Ni²⁺ and Cu²⁺, they are among the microelements that in small concentrations are used to perform vital functions in plants, such as electron transfer reactions, specific enzyme cofactors and constituents of the structure of several molecules (^{Pokorska et al., 2018}). An increase in the concentration of these metals would not only affect these processes, but, in addition, those associated with oxidative stress at the cellular level, caused by their high concentration. These results allow us to know what are the concentrations from which studies can be carried out on tolerance mechanisms in bean cultivation, since they would be those in which the crop survives without showing apparent damage. These are for the case of Pb²⁺ 100 ppm, for Ni²⁺ 5 ppm and for Cu²⁺ 20 ppm, assuming these are limits of phytotoxicity or crop tolerance.

Those findings are of great importance for current agriculture, mainly because in Cuba the planting of large-scale crops in soils that were unused was accelerated. This implies that, if the objective was to sow beans, a crop of national preference, without carrying out a study of the chemical-physical characteristics of the soil and it had high levels of bioavailable Pb²⁺, the risk of direct contamination to the man through the food chain would occur. In the case of Ni²⁺, the practical application would be different. Due to its low phytotoxicity limit in this crop, it could give rise to relatively simple bioassays that allow detecting areas at risk of being contaminated with this cation.

Evaluation of Changes in Membrane Permeability

The pH and electrical conductivity values of seedlings from germinated seeds in the presence of Pb²⁺ are presented in Figure 2, with variations observed in both indicators in most treatments with respect to control. Regarding the pH, the 25 ppm treatment started with a value greater than 7.4 and after 10 min it went down to find a relatively stable behavior around pH ꞊ 7 until 30 min. At concentrations of 10 ppm, it started with a pH value lower than 7. However, after 5 min, it rose to 7.6 and after 15 min, it reached stability at values close to pH ꞊ 7.4.

FIGURE 2 Values of pH and electrical conductivity in bean seedling leaves (Phaseolus vulgaris L.) variety CC-25-9-N with 15 DAG in the presence of different Pb²⁺ concentrations. 

For the concentrations of 50, 75, 100 and 125 ppm, pH values were started around neutrality, although the concentration of 50 ppm after 25 min rose until it reached its peak next to the 10 ppm treatment. The rest of the concentrations were maintained with constant pH values up to 30 min following a similar behavior to the control, although with values ​​slightly higher than the latter.

Although the cytoplasmic pH must be maintained above neutrality, there are changes that can affect this value. Plants when subjected to stress can cause extrusion or entry of H⁺ into the cellular interior. Small variations in pH are generally controlled by the buffer capacity of the cytoplasm, but this is a short-lived mechanism that is only maintained for 6-8 min (^{Budar and Roux, 2011}). Therefore, the plants of the present study would have to look for alternative routes of maintenance of membrane integrity, such as synthesizing or destroying organic acids for the control of pH variations, carrying out a physical-chemical mechanism of extrusion of H⁺ through the cytoplasm or a pumping of H⁺ to the vacuole through the tonoplast (^{Armbruster et al., 2017}).

In the case of electrical conductivity, the more specific behavior was produced by the concentration of 125 ppm that remained similar to that of the control treatment, although with lower values until 10 min, at which point it dropped sharply until a balance or stability was found at 20 min. The highest concentration of 125 ppm of Pb²⁺ was the one that produced the greatest modification in the cell membrane, since it induced a sharp change in the measurement of electrical conductivity until trying to find a homeostasis of the system.

This balance could be achieved from the exit or entry of H⁺, as well as the entrance of the metal into the cellular interior by means of pumps in the membranes. It has been proven that there are various specific pumps for toxic metal cations in the plants. Specific ATP-dependent enzymes act on these and have a structure similar to Na⁺/K⁺ pumps of plasma membranes (^{Li et al., 2016}). It is possible that, with the entrance of the metal into the cellular interior, structural changes or rearrangements in the composition of the membrane lipids were being carried out, which caused changes in the conductivity, behavior that is also revealed in the cation Ni²⁺ shown in Figure 3.

FIGURE 3 Values of pH and conductivity in bean seedling leaves (Phaseolus vulgaris L.) variety CC-25-9-N with 15 DAG in the presence of different Ni²⁺ concentrations. 

In the case of Ni²⁺, measurements could only be made in those treatments with sufficient plant material (concentrations of 1, 5 and 10 ppm). The concentration of 5 ppm began with a pH value near to 7, from this moment it began to decrease until 15 min, where it returned to take higher values around 6.8 trying to find a stabilization. The concentration of 10 ppm began with a pH value close to 7 and dropped sharply to 10 min, at which point a gradual rise was verified. Finally, the 1 ppm treatment, although it started with values higher than those of the control treatment, over time, it maintained a similar behavior to that. In the case of conductivity, all treatments were maintained with a behavior similar to that of the control treatment, although with lower conductivity values. This trend was also maintained for the case of Cu²⁺ shown in Figure 4.

FIGURE 4 Values of pH and conductivity in bean seedling leaves (Phaseolus vulgaris L.) variety CC-25-9-N with 15 DAG in the presence of different Cu²⁺ concentrations. 

In the seedlings that germinated with the Cu²⁺ solutions, the greatest differences between the treatments were caused in the pH measurement. For all concentrations of the metal under study, similarity was expressed regarding the general trend of variation as a function of time. Except for the concentration of 5 ppm that started with pH values higher than those of the control treatment and had a rapid decrease until 15 min, at which point an increase in its value began approaching that of the other concentrations used.

It is known that Cu²⁺ affects among other factors the transport of transmembrane ions, the concentration of intracellular Ca²⁺ and cell turgidity, as well as interfering with the transduction of cellular signals such as those of Ca²⁺ as a second messenger (^{Sun et al., 2010}). This metal could cause changes in transport in the cell by displacing Ca²⁺ from the membranes, and that leads to changes in intracellular pH. Another possible mechanism is the incorporation of Cu²⁺, once it is in the cytosol, to enzymes, amino acids or polypeptides such as phytokelatins, for a transport to the vacuolar interior, which would also cause a change in pH. It has been proven that Cu²⁺ induces changes in membrane permeability attributed to an increase in non-selective conductivity and to the inhibition or low activity of plasma membrane H⁺-ATPase pumps (^{Li et al., 2016}).

All those changes that affect the permeability of the membrane depend largely on its composition. The opening of the channels from transmembrane proteins and transduction signals, the transporters and the pumps, can be affected depending on the stress that is caused. By penetrating these systems, metal cations can cause the production of reactive oxygen species that are detected by receptors that activate protein kinases or signals in kinase cascades, changes in Ca²⁺ and calmodulin levels, regulation of gene expression and different transcription factors that would trigger, depending on the metal and its concentration, a specific response in the crop.

It could be considered that the bean variety CC-25-9-N shows a behavior of metal accumulator plant, which leads to establishing its potential in the possible negative effect for the trophic chain, not only because of the possibility of reaching the edible fruit, but because of the danger of using crop residues as a source of organic fertilizer. The results found present a great practical value for the precepts of agroecology and sustainable agriculture, which in turn, technically justify the systematic control of the content of metals in normative documents of agricultural pollution and Codex standards, based on the evaluation of risks organized by FAO and WHO.

Influence of Metal Cations on Antioxidant Systems

The POX and GPX enzyme activity values for the Pb²⁺, Ni²⁺ and Cu²⁺ cations at concentrations corresponding to the MLD and phytotoxicity limit or tolerance, are shown in Table 2.

TABLE 2 Activity of POX and GPX enzymes in bean seedling leaves exposed to different metal concentrations. 

Treatment	POX ∆470 min^-1 g^-1	GPX ∆420 min^-1 g^-1
Control	0,51^b	0,0019^c
Pb-100	0,64ª	0,0025^b
Pb-125	0,53^b	0,0033ª
Ni-5	0,13^d	0,0010^d
Ni-10	0,27^c	0,0017^c
Cu-20	0,10^d	0,0006^e
Cu-30	0,12^d	0,0009^de

a ... e: different letters represent means that differ significantly according to multiple range test (Tukey p<0.05)

Enzyme activity values indicate the removal of reactive oxygen species due to oxidative stress. The treatment corresponding to the concentration of 100 ppm of the Pb²⁺ cation showed higher values than the control, however, at a harmful concentration such as 125 ppm, values that do not differ from the control treatment were obtained, probably due to the activation of other antioxidant systems. The treatments corresponding to the microelements, in each of the concentrations studied, did not exceed the control values, presumably because they were incorporated into the plant's own metabolites or enzymes. The induction of “not so intense” stress could be used in signaling pathways to promote acceleration effects in the growth and development of seedlings, such as the synthesis of intermediary compounds and metabolites. It should be considered that germination was carried out under controlled conditions, not in a natural system where the own soil organic matter or the interaction with the microorganisms facilitated the absorption of the cations under study to the roots.

It is known that POX enzymes catalyze one of the final steps of lignin biosynthesis in roots (^{Kumar and Prasad, 2018}). In addition, GPX participates in metabolic reactions of lignin biosynthesis and AIA decomposition (^{Falade et al., 2018}). It is possible that these seedlings, and mainly those exposed to Pb²⁺, could activate as a defensive response the lignification of the root endodermis to prevent transport of the metal to the stem by keeping it out of the central cylinder, behavior demonstrated for metals such as Zn²⁺, Cu²⁺ and Cd²⁺ (^{Ledesma, 2014}).

GPX also catalyzes the oxidation of H⁺ donor chemical species due to the absence of a specific substrate, sometimes using guaiacol and sometimes ascorbate as a substrate for their reactions (^{Smirnoff, 2018}). Ascorbate is one of the precursors for the synthesis of phytokelatins, so if there is not enough of this peptide in the leaves to transport Pb²⁺ to the vacuoles, it is still potentially toxic and GPX would continue to eliminate the reactive species that generate inside the cell. It justifies the high values of this enzyme activity for that metal.

Changes in pH values and electrical conductivity, variables associated with membrane permeability, as well as POX and GPX activities depended on both, the metal and its concentration, so it is possible to infer some specificity in the plant - metal interaction, suggesting the predominance of different response mechanisms.

CONCLUSIONS

• For the bean (Phaseolus vulgaris L.) variety CC-25-9-N, the values of 100, 5 and 20 ppm as limits of phytotoxicity or tolerance to metals Pb²⁺, Ni²⁺ and Cu²⁺, respectively, are established.

• Changes in membrane permeability and antioxidant enzymatic activities depended on the metal and its concentration.

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⁸The mention of trademarks of specific equipment, instruments or materials is for identification purposes, there being no promotional commitment in relation to them, neither by the authors nor by the publisher.

Received: October 15, 2019; Accepted: March 13, 2020

^*Author for correspondence: Liane Portuondo Farías, e-mail: [e-mail]

Liane Portuondo Farías, Profesora Titular; Universidad Agraria de La Habana, Departamento de Química, Autopista Nacional y Carretera Tapaste km 23½ CP: 32700, Apartado Postal: 18-19, San José de las Lajas, Mayabeque, Cuba, e-mail: [e-mail]

Dariellys Martínez Balmori, Profesora Titular; Universidad Agraria de La Habana, Departamento de Química, Autopista Nacional y Carretera Tapaste km 23½ CP: 32700, Apartado Postal: 18-19, San José de las Lajas, Mayabeque, Cuba, e-mail: [e-mail]

Fernando Guridi Izquierdo, Profesor Titular; Universidad Agraria de La Habana, Departamento de Química, Autopista Nacional y Carretera Tapaste km 23½ CP: 32700, Apartado Postal: 18-19, San José de las Lajas, Mayabeque, Cuba, e-mail: [e-mail]

Alejandro Falcón Rodríguez, Investigador, Instituto Nacional de Ciencias Agrícolas. San José de las Lajas, Mayabeque, Cuba, e-mail: [e-mail]

Andrés Calderín García, Professor, Universidade Federal Rural do Rio de Janeiro, Laboratório de Solos, Rio de Janeiro, Brasil, e-mail: [e-mail]

João Paulo Machado Torres, professor, Universidade Federal do Rio de Janeiro, Laboratório de Radioisótopos, Rio de Janeiro. Brasil, e-mail: [e-mail]

The authors of this work declare no conflict of interests.