Isolation and expression analysis of partial sequences of heavy metal transporters from Brassica juncea by coupling high throughput cloning with a molecular fingerprinting technique

Abstract

Heavy metal transporters play a key role in regulating metal accumulation and transport in plants. These are important candidate genes to study in metal tolerant and accumulator plants for their potential use in environmental clean up. We coupled a degenerate primer-based RT-PCR approach with a molecular fingerprinting technique based on amplified rDNA restriction analysis (ARDRA) to identify novel ESTs corresponding to heavy metal transporters from metal accumulator Brassica juncea. We utilized this technique to clone several family members of natural resistance-associated macrophage proteins (NRAMP) and yellow stripe-like proteins (YSL) in a high throughput manner to distinguish between closely related isoforms and/or allelic variants from the allopolyploid B. juncea. Partial clones of 23 Brassica juncea NRAMPs and 27 YSLs were obtained with similarity to known Arabidopsis thaliana and Noccaea (Thlaspi) caerulescens NRAMP and YSL genes. The cloned transporters showed Brassica-specific changes in domains, which can have important functional consequences. Semi-quantitative RT-PCR-based expression analysis of chosen members indicated that even closely related isoforms/allelic variants of BjNRAMP and BjYSL have distinct tissue-specific and metal-dependent expressions which might be essential for adaptive fitness and heavy metal tolerance. Consistent to this, BjYSL6.1 and BjYSL5.8 were found to show elevated expressions specifically in cadmium-treated shoots and lead-treated roots of B. juncea, respectively.

Research on the Mechanisms of Plant Enrichment and Detoxification of Cadmium

“…If the YSL gene of maize is mutated, it will lead to the loss of part of the transport function of maize, which is manifested as leaf interveinal chlorosis, so it is named the “yellow stripe” transporter [ 73 ]. Das et al [ 74 ] cloned the BjYSL gene from the Cd hyperaccumulator, Brassica juncea , and BjYSL6.1 , which was specifically upregulated in shoots after Cd treatment, indicating that the YSL protein is involved in the transport of Cd from roots to shoots. Subsequently, Wang et al [ 75 ] overexpressed the BjYSL7 gene in tobacco and found that the Cd content in the BjYSL7 overexpression plants was significantly higher than that of the wild type, the roots were longer, and the root hair development was better, further proving that the BjYSL protein participates in the transport of Cd to shoots.…”

A new conceptual framework for plant responses to soil metals based on metal transporter kinetic parameters

“…intercellular transport to specialized cells [7,[9][10][11], or by excretion [12,13]. Homeostasis depends on metal ion transport from the apoplast to the symplasm, mediated by membrane proteins with transport functions, known as transporters, as the ions cannot move freely across the lipophilic cellular membranes [8,[13][14][15].…”

“…Titanium has been used most extensively as a soil contamination indicator for plant samples [48,53,54,57] because of its low concentration (less than 3 mg.kg -1 ) in plants and the large (10,000 to 500,000) soil : plant concentration ratio [43,56]. In addition, Cook et al [48] stated that Ti was considered a valid soil contamination indicator because there are three sources of Ti associated with plant samples, namely contamination during laboratory processing, vascular uptake via roots and field soil residue on shoot surfaces.…”

Effect of Heavy Metals in Plants of the Genus Brassica

“…Heavy metal transporters are very important in the regulation of metal transport and accumulation in plants, with consequences it their tolerance, and have thus been studied in different plant species including from the Brassica family [ 55 ]. Bhuiyan et al [ 10 ] generated a transgenic B. juncea plant, with the introduction of the AtATM3 gene, a member of the ATP-binding cassette (ABC) transporter family localized in the mitochondrial membrane of Arabidopsis thaliana .…”