Plant Molecular Biology 37: 87–97, 1998.
1998 Kluwer Academic Publishers. Printed in Belgium.
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
Holger J. Sch
afer, Angela Haag-Kerwer and Thomas Rausch
Botanisches Institut der Ruprecht-Karls-Universit
at, Im Neuenheimer Feld 360, 69120 Heidelberg, Germany
author for correspondence)
Received 5 August 1997; accepted in revised form 11 December 1997
Key words: Brassica juncea L., glutathione, phytochelatins, OAS(thiol)lyase,
glutathione synthetase, metallothionein 2, cadmium
In roots of Brassica juncea L. cadmium (Cd) exposure (25
M) 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),
-glutamylcysteine synthetase (
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,
-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 metallothioneincDNAs 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
-ECSmRNA showed a stronger increase. Expression analysis with 3
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
-ECS being most pronounced.
When Brassica juncea L. plants are exposed to cad-
mium (Cd), the heavy metal ions are taken up by the
rootsystem, and are, at least in part, translocated to the
shoot where they ﬁnally accumulate in the leaves [41,
42, 45]. A particularly strong accumulation has been
demonstrated for the trichomes covering the leaf sur-
face . The ability to efﬁciently translocate heavy
metal ions from the root to the shoot has led to the pro-
posal that B. juncea could be used to decontaminate
heavy metal-polluted soils .
At the cellular level higher plants may counter the
deleterious effects of heavy-metal ions by complexa-
tion with speciﬁc cysteine-rich polypeptides, known
as phytochelatins (PCs; [8–10, 24, 25, 31, 32]) and
metallothioneins (MTs; [26, 35, 52, 53]). PCs, the
ﬁrst group of heavy metal binding polypeptides, are
assembled enzymatically from GSH by PC synthetase
[8, 16, 18], the
-glutamylcysteinyl moiety being the
repeat unit (formular: [
X (X: Gly, Ser,
-Ala, or Glu)];  and references cited therein).
In vivo PCs of different chain length (
complexes with bound Cd and additional sulﬁde ions.
are thought to act as the transport vehicle from the
Cd complexes may represent the ﬁnal vacuolar stor-