Food crops are a major source of heavy metal intake in humans, which has prompted interest in understanding how plants take up, detoxify and retain heavy metals. Many human disorders have been attributed to the ingestion of heavy metals, including learning disabilities in children, dementia, impairment of bone metabolism and increased cancer rates ( Tong et al., 2000 Allen et al., 2002 Aschner and Walker, 2002 Ohta et al., 2002 Yu et al., 2002 Waisberg et al., 2003 Heck et al., 2009 Satarug et al., 2010).
Toxic metals such as lead, cadmium (Cd), mercury and the metalloid arsenic can accumulate in soils and water to levels that are detrimental to human and environmental health. Our results modify the glutathione-depletion driven model for sulfate assimilation gene induction during cadmium stress, and suggest that an enhanced oxidative state and depletion of upstream thiols, in addition to glutathione depletion, are necessary to induce the transcription of sulfate assimilation genes during early cadmium stress. Moreover, genetic, HPLC mass spectrometry, and gene expression analysis of the nrc1 and nrc2 mutants, revealed that intracellular glutathione depletion alone would be insufficient to induce gene expression of sulfate uptake and assimilation mechanisms. The nrc1 mutation was mapped to the γ-glutamylcysteine synthetase gene and the nrc2 mutation was identified as the first viable recessive mutant allele in the glutathione synthetase gene. Two nrc mutants, nrc1 and nrc2, were mapped, cloned and further characterized. The screen identified non-allelic mutant lines that fell into one of three categories: (i) super response to cadmium ( SRC) mutants (ii) constitutive response to cadmium ( CRC) mutants or (iii) non-response and reduced response to cadmium ( NRC) mutants.
Stably transformed luciferase reporter lines were ethyl methanesulfonate (EMS) mutagenized, and stable M 2 seedlings were screened for an abnormal luciferase response during exposure to cadmium.
The SULTR1 2 promoter (2.2 kb) was fused with the firefly luciferase reporter gene to quantitatively report the transcriptional response of plants exposed to cadmium. Microarray studies identified a high-affinity sulfate transporter ( SULTR1 2) among the most robust and rapid cadmium-inducible transcripts. To genetically elucidate cadmium-specific transcriptional responses in Arabidopsis, we designed a genetic screen based on the activation of a cadmium-inducible reporter gene. However, the mechanisms mediating toxic heavy metal-induced gene expression remain largely unknown. Plants exposed to heavy metals rapidly induce changes in gene expression that activate and enhance detoxification mechanisms, including toxic-metal chelation and the scavenging of reactive oxygen species.
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