cDNA cloning and expression analysis of genes encoding GSH synthesis in roots of the heavy-metal accumulator Brassica juncea L.: evidence for Cd-induction of a putative mitochondrial γ-glutamylcysteine synthetase isoform

cDNA cloning and expression analysis of genes encoding GSH synthesis in roots of the heavy-metal... In roots of Brassica juncea L. cadmium (Cd) exposure (25 µM) induces a massive formation of phytochelatins (PCs), which is accompanied by an only moderate decrease (−20%) of the putative PC precursor glutathione (GSH). As PC formation in roots could be the result of local GSH de novo synthesis and/or depend on GSH import from the shoot, we have analyzed the expression of the enzymes involved in GSH synthesis in the root, namely OAS(thiol)lyase (OAS-TL; catalysing the last step in Cys biosynthesis), γ-glutamylcysteine synthetase (γ-ECS), and glutathione synthetase (GSHS). cDNA clones were isolated from a cDNA library prepared from heavy metal exposed roots. Protein sequences from cDNA clones encoding OAS-TL, γ-ECS, and GSHS, all exhibited putative mitochondrial targeting sequences, however, for OAS-TL also two putative cytosolic isoforms were isolated. Furthermore, we have cloned several metallothionein cDNAs of the MT2 group. Northern blot analysis with coding region probes revealed that in roots of Cd-exposed plants transcript amounts for OAS-TL and GSHS were only moderately increased, whereas γ-ECS mRNA showed a stronger increase. Expression analysis with 3′-UTR probes indicated that among the putative mitochondrial OAS-TL, γ-ECS and GSHS isoforms only γ-ECS was up-regulated in response to Cd exposure. Conversely, transcripts for MT2 appeared to be slightly reduced. The results indicate that in roots Cd-induced PC synthesis correlates with a moderate increase of expression of genes involved in GSH synthesis, the change for γ-ECS being most pronounced. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

cDNA cloning and expression analysis of genes encoding GSH synthesis in roots of the heavy-metal accumulator Brassica juncea L.: evidence for Cd-induction of a putative mitochondrial γ-glutamylcysteine synthetase isoform

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Publisher
Springer Journals
Copyright
Copyright © 1998 by Kluwer Academic Publishers
Subject
Life Sciences; Biochemistry, general; Plant Sciences; Plant Pathology
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1023/A:1005929022061
Publisher site
See Article on Publisher Site

Abstract

In roots of Brassica juncea L. cadmium (Cd) exposure (25 µM) induces a massive formation of phytochelatins (PCs), which is accompanied by an only moderate decrease (−20%) of the putative PC precursor glutathione (GSH). As PC formation in roots could be the result of local GSH de novo synthesis and/or depend on GSH import from the shoot, we have analyzed the expression of the enzymes involved in GSH synthesis in the root, namely OAS(thiol)lyase (OAS-TL; catalysing the last step in Cys biosynthesis), γ-glutamylcysteine synthetase (γ-ECS), and glutathione synthetase (GSHS). cDNA clones were isolated from a cDNA library prepared from heavy metal exposed roots. Protein sequences from cDNA clones encoding OAS-TL, γ-ECS, and GSHS, all exhibited putative mitochondrial targeting sequences, however, for OAS-TL also two putative cytosolic isoforms were isolated. Furthermore, we have cloned several metallothionein cDNAs of the MT2 group. Northern blot analysis with coding region probes revealed that in roots of Cd-exposed plants transcript amounts for OAS-TL and GSHS were only moderately increased, whereas γ-ECS mRNA showed a stronger increase. Expression analysis with 3′-UTR probes indicated that among the putative mitochondrial OAS-TL, γ-ECS and GSHS isoforms only γ-ECS was up-regulated in response to Cd exposure. Conversely, transcripts for MT2 appeared to be slightly reduced. The results indicate that in roots Cd-induced PC synthesis correlates with a moderate increase of expression of genes involved in GSH synthesis, the change for γ-ECS being most pronounced.

Journal

Plant Molecular BiologySpringer Journals

Published: Oct 6, 2004

References

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