Impact of Some Toxic Metals on Important ABC Transporters in Soybean (Glycine max L.)

Abstract

In plants, ATP-binding cassette (ABC) transporters facilitate the movement of substrates across membranes using ATP for growth, development, and defense. Soils contaminated with toxic metals such as cadmium (Cd) and mercury (Hg) might adversely affect the metabolism of plants and humans. In this study, a phylogenetic relationship among soybeans' (Glycine max) ATP binding cassette (GmABCs) and other plant ABCs was analyzed using sequence information, gene structure, chromosomal distribution, and conserved motif-domain. The ontology of GmABCs indicated their active involvement in trans-membrane transport and ATPase activity. Thirty-day-old soybean plants were exposed to 100 μM CdCl₂ and 100 μM HgCl₂ for 10 days. Physiological and biochemical traits were altered under stress conditions. Compared to Control, GmABC transporter genes were differentially expressed in response to Cd and Hg. The qRT-PCR data showed upregulation of seven ABC transporter genes in response to Cd stress and three were downregulated. On the other hand, Hg stress upregulated four GmABC genes and downregulated six. It could be concluded that most of the ABCB and ABCG subfamily members were actively involved in heavy metal responses. Real-time expression studies suggest the function of specific ABC transporters in Cd and Hg stress response and are helpful in future research to develop stress-tolerant varieties of soybean.

Figure 1

Forty-days-old plants of soybean: (A) control plant, (B) Cd-treated plant (100 μM CdCl₂), and (C) Hg-treated plant (100 μM HgCl₂). It is noteworthy that first row showed histochemical detection of H₂O₂ in leaves, Cd accumulated more H₂O₂, second row denoted Cd and Hg treated leaves and third row indicated effect of Cd and Hg on fully grown plants in pots.

Figure 2

Conserved amino acids in 10 motifs of ABC transporter proteins in soybean with their width and length obtained through MEME 5.4.1 server (https://meme-suite.org).

Figure 3

Predicted three-dimensional structure of ABC transporter proteins of soybean obtained through Phyre2 server (http://www.sbg.bio.ic.ac.uk> phyre2) showing great diversity in their structural forms.

Figure 4

(A–C) GO analysis of GmABC transporter genes showing (A) biological functions, (B) cellular components, and (C) molecular functions. Photographs were developed through Gene Ontology Resource (http://geneontology.org).

Figure 5

Multivariate Data analysis of differentially expressed GmABC transporter genes developed by MetaboAnalyst 3.0 (https://www.metaboanalyst.ca) software showing (a) heatmap of differentially expressed GmABC transporter genes in the case of Control or heavy metal treated (C1, C2, and C3 as Control, Hg1, Hg2, and Hg3 as mercury treated, and Cd1, Cd2, and Cd3 as cadmium treated condition), (b) loadings plot, (c) VIP plot, and (d) biplot.

Figure 6

(A) 1% Agarose gel showing isolated RNA bands of Control and stress (Cd and Hg)-treated plant with 500 bp DNA ladder, (B) PCR amplified products of GmABC transporter genes in 2% agarose gel (lanes 1–10). Membrane edges of gels were not visible due to cropping of gels as both the RNA isolation and RT-PCR bands run on the same gel simultaneously due to the lack of time constraints. Lane description; (1) PCR amplicon of control V/s GmABC5, (2) PCR amplicon of control V/s GmABC4, (3) PCR amplicon of control V/s GmABC8, (4) PCR amplicon of control V/s GmABC7, (5) PCR amplicon of control V/s GmABC9, (6) PCR amplicon of control V/s GmABC3, (7) PCR amplicon of control V/s GmABC10, (8) PCR amplicon of control V/s GmABC6, (9) PCR amplicon of control V/s GmABC2, and (10) PCR amplicon of control V/s GmABC1, L- 100 bp DNA ladder, and actin gene as an internal control.

Figure 7

Relative gene expression profiles of soybean ABC transporter genes (GmABC) in control (-Cd,-Hg) and in response to Cadmium (100 μM) and Mercury (100 μM) treatments using qRT-PCR in which actin is used as an internal control. Asterisks indicate p < 0.05 (t-test). All data are represented as Mean ± standard error (SE). The measurements were made using three independent biological replicates.

Figure 8

Venn representation of expression pattern of ABC transporter genes of soybean in response to Cd and Hg stress treatment.

Figure 9

Phylogenetic tree of important ABC transporter proteins developed using MEGA5.04 (https://www.megasoftware.net) from soybean and other plant species. Blue circle dots represent-ABC transporter genes in Glycine max including (GmABCs in square dots), brown: ABC transporter genes, yellow: Mucuna pruriens ABC transporter genes, pink: Oryza sativajaponica ABC transporter genes, red: Capsicum annuum ABC transporters, orange: Theobroma cacao ABCs, dark green: Zea mays, light green: Vitis vinifera, and cream: Cucumis sativus ABC transporter genes.

Figure 10

Detoxification of heavy metal Cd and Hg in soybean plant cells which involved ABC transporter genes. PC: phytochelatin, PCS: phytochelatin synthase, red dots: Cd ions, and green dots: Hg ions. Seven ABC transporter genes were located on the plasma membrane with two located on the membrane of the vacuole and a single gene located in the cytoplasm of the plant cell.