Characterization of Brassica rapa metallothionein and phytochelatin synthase genes potentially involved in heavy metal detoxification

Abstract

Brassica rapa is an important leafy vegetable that can potentially accumulate high concentrations of cadmium (Cd), posing a risk to human health. The aim of the present study was to identify cadmium detoxifying molecular mechanisms in B. rapa using a functional cloning strategy. A cDNA library constructed from roots of B. rapa plants treated with Cd was transformed into the Cd sensitive yeast mutant strain DTY167 that lacks the yeast cadmium factor (YCF1), and resistant yeast clones were selected on Cd containing media. Two hundred genes potentially conferring cadmium resistance were rescued from the surviving yeast clones and sequenced. Sequencing analysis revealed that genes encoding for metallothionein (MT)1, MT2a, MT2b and MT3, and phytochelatin synthase (PCS)1 and PCS2 accounted for 35.5%, 28.5%, 4%, 11.3%, 18.7% and 2%, respectively of the genes identified. MTs and PCSs expressing DTY167 cells showed resistance to Cd as well as to Zn. PCS1 expressing yeast cells were also more resistant to Pb compared to those expressing MTs or PCS2. RT-PCR results showed that Cd treatment strongly induced the expression levels of MTs in the root and shoot. Furthermore, the different MTs and PCSs exhibited tissue specific expression. The results indicate that MTs and PCS genes potentially play a central role in detoxifying Cd and other toxic metals in B. rapa.

Fig 1. Screening of Cd tolerance gene in Brassica rapa.

(A) Insert size of B. rapa cDNA library. Inserts of the B, rapa cDNA library in the pYES2 vector were amplified using PCR with vector linker primers, and electrophoresed in 1% agarose gel. (B) First selection of Cd tolerance genes in B. rapa. The Cd-sensitive yeast mutant (DTY167) was transformed with a cDNA library of B. rapa and grown on synthetic galactose (SG) agar plates supplemented with 70 μM CdCl₂ to select clones harboring Cd tolerance genes. (C, D) Second selection of Cd tolerance genes in B. rapa. The surviving clones from the 1’st screening were grown on synthetic dextrose (SD) and synthetic galactose (SG) media supplemented with 40 μM CdCl₂ to select real positive clones. False-positive clones surviving on SD supplemented with CdCl₂ were discarded. The clones growing on SG but not on SD were further analyzed.

Fig 2. Relative frequency of Cd tolerance genes and phylogenetic trees of BrMTs in Brassica family.

(A) Relative frequency of Cd tolerance genes in B. rapa. Cd tolerance genes were identified from 200 yeast clones exhibiting Cd tolerance, and the relative frequency of Cd tolerance genes was analyzed. (B-D) Phylogenetic analysis using the neighbor-joining method implemented in MEGA X. The results are based on multiple alignments of MTs amino acid sequences. The bootstrap values (percentage) of 1000 replicates are shown at the branching points.

Fig 3. Tissue specific expression of BrMTs and BrPCSs in B. rapa.

To analyze the expression of B. rapa Cd tolerance genes (BrMTs and BrPCSs), qRT-PCR was performed with cDNA synthesized from total RNA as a template and gene-specific primers (S1 Table). The expression levels of Cd tolerance genes were normalized to the level of actin, and the values indicate the average and standard error (biological replicates: 3). Different letters indicate that the means (between various samples) are significantly different by the Tukey HSD test (P≤0.05). R, roots from one-month old plants; RL, rosette leaves from one-month old plants; Fl, flower; AL, leaf from flowering plants; St, stem from flowering plants.

Fig 4. Cd-inducible expression of BrMTs and BrPCSs in B. rapa.

One-month old B. rapa plants were treated with 100 μM CdCl₂ for different times and harvested to analyze the expression levels of B. rapa Cd tolerance genes. qRT-PCRs were performed with cDNA synthesized from the RNA as a template and gene-specific primers (S1 Table). The expression levels of Cd tolerance genes were normalized to the level of actin, and the values indicate the average and standard error (biological replicates: 3). *P < 0.05, **P < 0.01 (Student’s t-test).

Fig 5. Enhanced heavy metal tolerance in yeast strains expressing B. rapa Cd tolerance genes.

DTY167 yeast cells were transformed with empty vector (V), BrMT1a, BrMT1b, BrMT1c, BrMT2a, BrMT2b, BrMT3, BrPCS1 and BrPCS2. The yeast strains were cultured in synthetic dextrose without uracil (SD ura−) liquid medium, spotted on synthetic galactose without uracil (SG ura−) agar plates supplemented with 40 μM CdCl₂, 5 mM ZnSO₄ or 1.2 mM Pb-tartrate, and cultured at 30 °C for 3–4 d.

Fig 6. Analysis of Zn binding amino acid residues in B. rapa MTs.

Zn binding amino acid residues of BrMT1a, BrMT2a, BrMT2b and BrMT3 were identified through the ZincBinder (http://proteininformatics.org/mkumar/znbinder/), and alignment of BrMTs was performed using CLUSTALW program of BioEdit. (A) Zn binding amino acid positions and Zn binding scores of BrMT1a, BrMT2a, BrMT2b and BrMT3. (B) Alignment of BrMT1a, BrMT2a, BrMT2b and BrMT3 protein. Boxes indicate putative Zn binding amino acid residues.