Quantitative Estimation of Synergistic Toxicity of Cu and Zn on Growth of Arabidopsis thaliana by Isobolographic Method

Abstract

Heavy metal is one of the most frequent soil contaminants and contaminated soils generally include numerous metals. Although exposure to multiple metals may increase the toxicity to humans and ecosystems, only additive effects are considered in the risk assessment. In this study, the synergistic effect of heavy metals (Cu and Zn) on a model plant, Arabidopsis thaliana, was quantified by the isobolographic method. The plant was cultured via the growth assay method on a plant agar containing individual heavy metals or combinations of Cu + Zn in a growth chamber. The concentration of Cu varied by eight levels from 0 to 200 μM and the concentration of Zn also varied by eight levels from 0 to 400 μM. In the combination of metals, each of the three levels of Cu (25–75 μM) and Zn (20–100 μM) were applied. After 8 days, plants were harvested for root/shoot weight and measured for leaf chlorophyll and carotenoid content. The primary and secondary root elongation of A. thaliana was estimated using image analysis to calculate total root length. The EC50 values of Cu and Zn on A. thaliana, based on the total root length, were 40.0 and 76.4 μM, respectively. When two heavy metals were administered in combination, the EC values decreased less than those of the individual metals. The average value of the combination index was 0.6, proving the synergistic toxic effect on the root growth of A. Thaliana. As a result, the isobolograhic method is a useful tool for estimating the quantitative toxic effect of chemicals on plants.

Copper and Zinc Accumulation in Young Leaves of Eruca sativa (L.) Grown in Soilless Culture

“…The metal concentration causing toxicity in plants depends on the duration of exposure and the plant species, and different threshold levels for Cu or Zn have been reported in the literature. For example, in the agar-grown seedlings of Arabidopsis thaliana after eight days of treatment, Cu and Zn concentrations of 40.0 and 76.4 µM, respectively, reduced the root growth by 50% [6]. Concentrations up to 20 µM of Zn have been used for rocket microgreens [30], whereas rocket plants biofortified with 80 µM of Zn were used to evaluate the bioaccessibility of the element after in vitro gastrointestinal digestion [16].…”

“…In plants, heavy metals can cause physiological and morphological modifications by inducing the production of reactive oxygen species (ROS), inhibiting enzyme activity, reducing photosynthesis, and activating detoxification mechanisms [6]. However, some heavy metals, including copper (Cu) and zinc (Zn), are also essential trace elements (or micronutrients) involved in biochemical reactions or in functional molecules, such as enzymes or structural proteins [7].…”

“…Copper is a cofactor in enzymes that regulate photosynthesis and activate ROS scavenging. Along with the cell damage caused by ROS production, Cu poisoning causes physiological and morphological alterations, such as limited plant growth and photosynthetic activity, chlorosis, and a reduced nutrient content in root tissues [6]. Similarly, Zn is a widespread pollutant and is the second most abundant transition metal in living organisms after iron [13].…”

“…Zn is involved in enzyme activation, photosynthesis, nitrogen (N) metabolism, and cell multiplication [14]. Excess Zn can be toxic to plants, with effects similar to those caused by excess Cu: chlorosis, the deficiency of other micronutrients, the inactivation of enzymes involved in ROS scavenging (e.g., peroxidase), and the inhibition of root development [6]. It has been reported that Zn can cause toxicity in plants at tissue concentrations above 300 µg/g of dry matter [10], provoking nutritional and photosynthetic impairment.…”

Arbuscular mycorrhiza mitigates zinc stress on Eucalyptus grandis through regulating metal tolerance protein gene expression and ionome uptake

“…Zinc (Zn) was an utmost important micronutrient for all living organisms, and it acted as catalytic and structural component in a large number of enzymes and regulatory proteins ( Maret, 2009 ; Zhang et al., 2018 ; Kaur and Garg, 2021 ; Bae et al., 2022 ). Zn played an important role in regulating plant growth and development, which involves modulating a wide range of physiological processes: cell proliferation, respiration, auxin biosynthesis, and antioxidative defenses ( Broadley et al., 2007 ; Zhang et al., 2018 ; Kaur and Garg, 2021 ; Bae et al., 2022 ). However, high concentration of Zn can be toxic.…”