title: Feedback inhibition by thiols outranks glutathione depletion: a luciferase-based screen reveals glutathione-deficient γ-ECS and glutathione synthetase mutants impaired in cadmium-induced sulfate assimilation.
abstract: 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. However, the mechanisms mediating toxic heavy metal-induced gene expression remain largely unknown. To genetically elucidate cadmium-specific transcriptional responses in Arabidopsis, we designed a genetic screen based on the activation of a cadmium-inducible reporter gene. Microarray studies identified a high-affinity sulfate transporter (SULTR1;2) among the most robust and rapid cadmium-inducible transcripts. 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. 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 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. Two nrc mutants, nrc1 and nrc2, were mapped, cloned and further characterized. 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. 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. 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.
authors.affiliation: Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, California 92093-0116, USA.
authors.forename: Timothy O
authors.initials: TO
authors.lastname: Jobe
authors.forename: Dong-Yul
authors.initials: DY
authors.lastname: Sung
authors.forename: Garo
authors.initials: G
authors.lastname: Akmakjian
authors.forename: Allis
authors.initials: A
authors.lastname: Pham
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Komives
authors.forename: David G
authors.initials: DG
authors.lastname: Mendoza-Cózatl
authors.forename: Julian I
authors.initials: JI
authors.lastname: Schroeder
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism; D000483:Alleles; D027321:Anion Transport Proteins / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D002104:Cadmium / Q000494:pharmacology*; D032461:Chromosomes, Plant / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004790:Enzyme Induction; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D025461:Feedback, Physiological; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005816:Genetic Complementation Test; D005978:Glutathione / Q000378:metabolism*; D005981:Glutathione Synthase / Q000235:genetics / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010084:Oxidation-Reduction; D020161:Physical Chromosome Mapping / Q000379:methods; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D017382:Reactive Oxygen Species / Q000494:pharmacology; D036226:Seedlings / Q000187:drug effects / Q000201:enzymology; D013431:Sulfates / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D027321:Anion Transport Proteins; D029681:Arabidopsis Proteins; D017382:Reactive Oxygen Species; D013431:Sulfates; D013438:Sulfhydryl Compounds; C455459:Sultr1;1 protein, Arabidopsis; D002104:Cadmium; D005020:Ethyl Methanesulfonate; D008156:Luciferases; D005981:Glutathione Synthase; D005978:Glutathione