Journal of Experimental Botany, Vol. 56, No. 421, pp.

30173027, November 2005

doi:10.1093/jxb/eri299 Advance Access publication 10 October, 2005

RESEARCH PAPER

Identication of cadmium-regulated genes by cDNA-AFLP

in the heavy metal accumulator Brassica juncea L.
Nicola Fusco, Lorenza Micheletto, Giovanni Dal Corso, Lorena Borgato and Antonella Furini*
University of Verona, Department of Science and Technology, Strada le Grazie 15, I-37134 Verona, Italy
Received 7 June 2005; Accepted 25 August 2005

Introduction

In this study, cDNA-amplied fragment length polymorphism (cDNA-AFLP) analysis was employed to
identify genes that exhibited a modulated expression
following cadmium (Cd) treatment in Brassica juncea
grown in hydroponic culture. Plants were treated
for 6 h, 24 h, and 6 weeks with 10 lM Cd(NO3)2 and
untreated 6-week-old plants were used as controls.
Cd content was measured at these four time points.
Long exposure to Cd affected root morphology: roots
appeared thinner and sent out side roots. Seventythree transcript-derived fragments were identied as
Cd responsive. Fifty-two of them showed signicant
homology to genes with known or putative function,
10 transcript-derived fragments were homologous to
uncharacterized genes, while 11 transcript-derived
fragments did not show signicant matches. The
expression pattern of several of these genes was
conrmed by northern blot analysis. Fifty-two genes
of known or putative function were transcriptional
factors, expression regulators, and stress responding
and transport facilitation genes, as well as genes
involved in cellular metabolism and organization and
the photosynthetic process, suggesting that a multitude of processes are implicated in Cd stress response. The transcription of drought- and abscisic
acid-responsive genes observed in this study also
suggested that Cd imposes water stress and that
abscisic acid may be involved in the Cd plant
response.

Environmental pollution by toxic metals has accelerated

dramatically since the beginning of the industrial revolution
(Nriagu, 1979). Cadmium, a non-essential heavy metal, is
considered one of the major pollutants (Alloway and
Steinnes, 1999). It is widespread in soil containing waste
materials from zinc mines, in sewage water used for
irrigation, in sludge-amended soils, and in soil fertilized
with cadmium-rich phosphate fertilizers (Huttermann et al.,
1999). Cadmium is also released into the environment by
urban traffic, heating systems, and waste incinerators
(Sanita` di Toppi and Gabbrielli, 1999). Toxicity to living
cells is caused by very low cadmium concentrations and
its presence in the food chain can be highly dangerous as it
can cause significant damage to the human body (Buchet
et al., 1990) and is a suspected carcinogen (Vido, 2001).
Although Cd is a non-essential element for plant mineral
nutrition it is taken up easily by roots and transported
through the xylem to the vegetative and reproductive
organs, thus affecting nutrient uptake and homeostasis
and inhibiting root and shoot growth and yield production
(Sanita` di Toppi and Gabbrielli, 1999; Metwally et al.,
2005). However, certain plant species have the ability to
survive and reproduce on soils containing high concentrations of metals in forms that are toxic or inimical to other
plants (Macnair and Baker, 1994). The ability of these
plants to survive on metal-polluted soils is not only due
to their capacity to take up, translocate, and sequester
metals, but is also based on mechanisms that allow them
to tolerate high levels of the element in root and shoot cells
by alleviating their toxic effects (Salt et al., 1998). Tolerance is achieved by internal detoxification and probably
involves cell compartmentation and metal complexation.
For several metals and species, genetic analysis has demonstrated that tolerance is controlled by a small number
of major genes, with additional modifiers determining the

Key words: Brassica juncea, cadmium, cDNA-AFLP, gene

expression, heavy metals.

* To whom correspondence should be addressed. Fax: +39 045 8027950. E-mail: antonella.furini@univr.it
Published by Oxford University Press [2005] on behalf of the Society for Experimental Biology.

Downloaded from http://jxb.oxfordjournals.org/ by guest on August 8, 2014

Abstract

3018 Fusco et al.

Materials and methods

Plant material, culture conditions, and Cd treatment
Brassica juncea (L.) Czern, cv. Aurea, was selected because previous
work screening 10 cultivars (Bona et al., unpublished results) had
demonstrated its enhanced ability to accumulate Cd from hydroponic
solution into the above-ground (harvestable) parts. Seeds were rinsed
with distilled water and incubated on soaked 3MM Whatman paper
(Whatman, Maidstone, UK) in Petri dishes at 20 8C for 4 d.
Germinated seeds were mixed with sand and vermiculite (1:1) and
transferred to holes (1.5 cm diameter) on polyethylene discs used as
floating supports. Plants were grown for 6 weeks in continuously
aerated hydroponic nutrient solution. Each pot held three plants and
1.0 l of half-strength Hoagland solution (Hoagland and Arnon, 1938)
with pH adjusted to 5.7. The plants were maintained in greenhouse
conditions, and the nutrient solution changed every 57 d depending
on evaporative demand. To identify the gene transcriptional changes

modulated by Cd, 6-week-old plants were treated for 6 h and 24 h

with 10 lM Cd(NO3)2 while other plants had received the same Cdtreatment from seed germination for 6 weeks. Untreated 6-week-old
plants were used as controls. Plants were then harvested. Some of the
samples were quickly frozen in liquid nitrogen and stored at 80 8C
for RNA extraction, while others were analysed for Cd content.
Determination of Cd content
For the assay of Cd concentration, plants were harvested, rinsed in
distilled water, weighed, and oven-dried at 85 8C for 36 h. Dried
samples were homogenized before analysis using a Wiley mill.
Cd analysis was performed after microwave-assisted acid digestion
(EPA 3052, 1996) by means of ICP-MS analysis (EPA 200.8).
mRNA isolation and cDNA synthesis
Total RNA was extracted from plants and sampled after the indicated
treatments, using Trizol reagent (Gibco, Germany). Total RNA
concentration was determined spectrophotometrically and adjusted
to a final concentration of 1 lg ll1. Poly (A)+ RNA fractions were
isolated from 1 mg total RNA with the Oligotex mRNA Minikit
(QIAGEN, Germany). First and second cDNA strands were synthesized according to standard protocols (Sambrook et al., 1989).
cDNA-AFLP analysis
The cDNA-AFLP-based transcript profiling procedure was performed according to the method described in Breyne et al. (2002).
Double-stranded cDNA (500 ng) was used for cDNA-AFLP analysis.
The restriction enzymes used were BstYI and MseI (New England
Biolabs, Beverly, MA, USA). For pre-amplification, an MseI-primer
without a selective nucleotide was combined with a BstYI-primer
containing a T or a C at the 39 end. The amplification mixtures
obtained were diluted 600-fold and 5 ll were used for final selective
amplifications according to Breyne et al. (2002). BstT- and MseIprimers and BstC- and MseI-primers with one selective nucleotide,
respectively, were used for the cDNA-AFLP analysis, and all 32
possible primer combinations were performed. Selective [33P]ATPlabelled amplification products were separated on a 6% polyacrylamide gel run at 1100 V for 3 h. Gels were dried onto 3MM Whatman
paper, and positionally marked before being exposed to Kodak
Biomax film (Amersham, Pharmacia, USA) for 2 d.
Isolation and sequencing of fragments
Films were aligned with markings on the gels. The bands of interest
were marked, cut out with a razor blade, and incubated in 100 ll of
water at 65 8C for 15 min and then left overnight at room temperature
for DNA elution. The eluted DNA was re-amplified using the same
PCR conditions and primer combination as for the selective amplification. The re-amplified products representing the Cd-regulated
transcript-derived fragments (TDFs) were checked on 2% agarose gel
and directly sequenced using the selective BstYI-primer as a sequencing primer. TDFs that failed to be sequenced were ligated to the
pGEM-T EASY vector (Promega, Southampton, UK) and clones
were then sequenced using the T7 or SP6 primer. Database searches
were performed using the BLAST Network service [NCBI (National
Center for Biotechnology Information); http://www.ncbi.nlm.nih.
gov/BLAST]. The sequence of each TDF was searched against all
sequences in the databases using the BLASTN and BLASTX
programs (Altschul et al., 1997).
Northern blot analysis
Poly (A)+ RNA was prepared for Cd-treated as well as untreated
plants by chromatography on oligo dT-cellulose (Bartels and
Thompson, 1983). Three micrograms of mRNA per lane were
fractionated on a 1% denaturing formaldehyde/agarose gel and transferred onto a positively charged nylon membrane. The procedures

Downloaded from http://jxb.oxfordjournals.org/ by guest on August 8, 2014

level of tolerance (Smith and Macnair, 1998; van Hoof

et al., 2001)
Extensive studies have characterized the mechanisms
underlying metal accumulation and tolerance in plants (for
reviews, see Clemens, 2001; Schutzendubel and Polle,
2002) and several plant genes that regulate the adaptive
response to heavy metal-contaminated soil have been
identified. For instance, it is known that phytochelatins
(PCs) play a major role in heavy-metal detoxification.
Indeed, metals bound to PCs are probably transported into
the vacuoles (Cobbett, 2000). Studies of mutants and transgenic plants corroborated the importance of PCs for protection from heavy metals and lack of PC synthase activity led
to increased sensitivity (Howden et al., 1995; Zhu et al.,
1999). However, most molecular components of the signal
transduction pathway involved in gene regulation are as
yet unidentified. cDNA-amplified fragment length polymorphism (cDNA-AFLP) is an efficient, sensitive, and
reproducible technology for the isolation of differentially
expressed genes (Bachem et al., 1996); it does not require
prior sequence information and is therefore a useful tool for
the identification of novel genes (Ditt et al., 2001).
In this study, cDNA-AFLP was employed to identify
genes that exhibited a modulated expression following Cdtreatment in Brassica juncea. This species was chosen
because it is characterized by rapid growth, high biomass,
and an appreciable capacity to take up Cd as well as other
toxic metals (Kumar et al., 1995; Rugh, 2004). In particular, Cd appeared to accumulate preferentially in roots, part
of it is translocated from root to shoot, where it finally
accumulates in the leaves (Salt et al., 1995a, b; Clemens
et al., 2002). High accumulation was also demonstrated for
the trichomes covering the leaf surface (Salt et al., 1995a),
and it has been reported that B. juncea seedlings grown in
aquaculture were able to accumulate and remove Cd from
contaminated water (Salt et al., 1997). Further characterization of the Cd-responsive genes isolated in this work,
may be helpful for a better understanding of the mechanisms of Cd accumulation and tolerance in plants.

Cadmium-modulated genes in B. juncea

for radiolabelling and hybridization were performed at 42 8C as
described previously (Sambrook et al., 1989). Filters were washed
three times for 10 min at 65 8C with 23 SSC and 0.1% SDS. To check
that the RNAs were equally loaded, the filters were re-probed with
a barley ubiquitin cDNA clone (Gausing and Barkardottir, 1986).

Results
Plant growth and Cd accumulation

times to Cd (6 h, 24 h, and 6 weeks) were chosen to detect

genes rapidly responding to Cd shock, and genes whose
expression is modulated by the continuous presence of Cd
in the culture medium. By using 32 primer combinations,
about 3000 cDNA fragments were counted and all bands
longer than 80 bp in length were compared in the four tested
Cd conditions. About 100 up-regulated or down-regulated
gene fragments were identified as Cd modulated. These
Cd-regulated TDFs, varying in length from 80 to 500 bp,
were excised from the gels, re-amplified by PCR, and sequenced. Only in a few cases did different cDNA fragments
belong to the same transcript; in addition, sequencing failed
for several TDFs even after cloning. These fragments were
not characterized further.
TDF sequences were compared with those present in the
GenBank database (Table 1). The clones corresponding to
different TDFs were renamed as BjCdR (B. juncea Cdregulated). Of the total 73 TDFs sequenced (Table 1), 52
(71.2%) showed significant homology to genes with known
or putative function, while 10 TDFs (13.7%) were homologous to uncharacterized genes (EST and unknown proteins). Of the remaining 11 TDFs, eight (11.0%) did not
show significant matches; they may represent yet uncharacterized genes or the TDFs sequenced represent the 39 end

Analysis of cDNA-AFLP
cDNA-AFLP analysis was performed to identify the genes
responsive to Cd in B. juncea (Fig. 2). Different exposure

Fig. 1. Cd contents of plants maintained under greenhouse conditions,

grown for 6 weeks in hydroponic solution and exposed to 10 lM
Cd(NO3)2 for different times. 0, 6 h, 24 h, and 6 w (weeks) represent the
exposure times to Cd.

Fig. 2. cDNA-AFLP autoradiography showing the TDFs induced or

repressed by Cd treatment. 0, 6 h, 24 h, and 6 w (weeks) represent the
exposure times to Cd.

Downloaded from http://jxb.oxfordjournals.org/ by guest on August 8, 2014

Plants of B. juncea cv. Aurea, grown for 6 weeks with 10

lM Cd(NO3)2 present in the nutrient solution, exhibited
growth inhibition of both roots and shoots, with roots being
more affected by Cd than shoots. At the end of the experimental period leaves of these plants showed symptoms of
chlorosis. The decrease in root elongation was also accompanied by changes in morphology. Roots became thinner
and were putting out numerous side roots.
Control plants maintained under greenhouse conditions
and grown for 6 weeks in hydroponic solution, without Cd,
showed a concentration of 15.29 lg kg1 in their dry biomass. After exposure to 10 lM Cd(NO3)2, for either 6 h or
24 h, Cd concentration increased. Under these conditions,
the amount of Cd in the dry biomass was 70.59 lg kg1
DW and 123.53 lg kg1 DW at 6 h and 24 h, respectively,
confirming the adsorption even after a short time of Cd
exposure. Plants exposed for 6 weeks to 10 lM Cd(NO3)2
showed an accumulation of 805.88 lg kg1 DW (Fig. 1). It
is worth noting the accumulation capacity of B. juncea in
hydroponic culture (0.08% of dry weight), although the
severe growth impairment and stressed condition of these
plants make a meaningful comparison between Cd content
and gene expression difficult.

3019

3020 Fusco et al.

Table 1. Homologies of sequences of cDNA-AFLP fragments to sequences in the databases
TDF

Length
(bp)

Accession
number

Homologya

BLAST
scoreb,c

Expression
patternd

BjCdR1
BjCdR2
BjCdR3
BjCdR4
BjCdR5
BjCdR6
BjCdR7
BjCdR8
BjCdR9
BjCdR10
BjCdR11
BjCdR12

110
120
102
355
161
205
247
231
113
379
130
86

DT317657
DT317658
DT317659
DT317660
DT317661
DT317662
DT317663
DT317664
DT317665
DT317666
DT317667
DT317668

2e-12
8e-09
2e-07
2e-24
3e-04
1e-14
3e-29
2e-15
2e-10*
3e-47
4e-12*
3e-07

0
6 h,
6 h,
0
6 h,
24 h
6h
6h
6h
6 h,
24 h
6h

BjCdR13
BjCdR14
BjCdR15

176
151
288

DT317669
DT317670
DT317671

9e-08*
3e-08
1e-11

0
6h
6h

BjCdR16

426

DT317672

2e-53

6 h, 24 h

BjCdR17
BjCdR18
BjCdR19

302
306
331

DT317673
DT317674
DT317675

7e-21
6e-36
3e-25

6h
0
6h

BjCdR20

381

DT317676

BjCdR21

392

DT317677

BjCdR22

168

DT317729

BjCdR23
BjCdR24
BjCdR25
BjCdR26

150
310
287
86

DT317678
DT317679
DT317680
DT317681

BjCdR27
BjCdR28

135
127

DT317682
DT317683

BjCdR29

321

DT317684

BjCdR30
BjCdR31

386
287

DT317685
DT317686

BjCdR32
BjCdR33
BjCdR34

236
321
192

DT317687
DT317688
DT317689

BjCdR35

174

DT317690

BjCdR36
BjCdR37

485
355

DT317691
DT317692

BjCdR38
BjCdR39
BjCdR40

123
202
141

DT317693
DT317694
DT317695

BjCdR41

309

DT317696

BjCdR42
BjCdR43
BjCdR44

133
123
225

DT317697
DT317698
DT317699

BjCdR45
BjCdR46
BjCdR47
BjCdR48

240
153
115
152

DT317700
DT317701
DT317702
DT317703

Expressed protein from A. thaliana (NP_196620.1)

Expressed protein from A. thaliana (NP_181834.1)
Expressed protein from A. thaliana (NP_850785.1)
Expressed protein from A. thaliana (NP_565726.1)
Unknown protein from A. thaliana (AAM63312.1)
Expressed protein from A. thaliana (NP_566625.1)
Expressed protein from A. thaliana (NP_565922.1)
Expressed protein from A. thaliana (NP_566847.1)
Putative protein mRNA from A. thaliana (AY128790.1)
Expressed protein from A. thaliana (NP_174692.1)
ABC1 family protein mRNA from A. thaliana (NM_180205)
MYB family transcription factor (MYB59) from A. thaliana
(NP_851226.1)
Putative initiation factor 5A mRNA A. thaliana (AF492850.1)
Transcription factor GBF5 from A. thaliana (AAG17474.1)
bZIP family transcription factor (TGA3) from A. thaliana
(NP_564156.1)
Zinc-finger (B-box type) family protein/salt tolerance protein
(STO) from A. thaliana (NP_172094.1)
CP12 protein, chloroplast precursor from P. sativum (T06562)
Photosystem II family protein from A. thaliana (NP_563687.1)
Photosystem I reaction centre subunit VI, chloroplast precursor
(PSI-H) (light harvesting complex I 11 kDa protein) from B. rapa
(O04006)
Chorismate mutase, chloroplast precursor (CM1) from A. thaliana
(P42738)
Chlorophyll a-b binding protein 1, chloroplast precursor (LCHII
type I CAB-1) (LCHP) from S. alba (P13851)
Putative cytochrome P450 protein mRNA from A. thaliana
(AY050890)
LHCI type II from L. temulentum (CAA55864)
ELIP from B. rapa subsp. pekinensis (AAR11456)
PSI-H subunit mRNA from B. rapa (U92504)
Glutamine synthetase, chloroplast precursor (glutamateammonia
ligase) (GS2) from B. napus (Q42624)
Glutamine-synthetase mRNA from A. thaliana (AY059932.1)
Cysteine synthase (O-acetylserine sulfhydrylase) (O-acetylserine
(thiol)-lyase) from B. juncea (O23735)
b-Hydroxyacyl-ACP dehydratase, putative from A. thaliana
(NP_196578.1)
Putative histone deacetylase from A. thaliana (AAG28473.1)
Ubiquitin-conjugating enzyme E2-17 kDa (ubiquitin-protein ligase)
(ubiquitin carrier protein) from L. esculentum (P35135)
Starch excess protein (SEX1) from A. thaliana (NP_563877.1)
AAA-type ATPase family protein from A. thaliana (NP_182074.2)
NADH-ubiquinone oxireductase-related from A. thaliana
(NP_566608.1)
PBS lyase HEAT-like repeat-containing protein from A. thaliana
(NP_197483.1)
Phenylalanine ammonia lyase from A. thaliana (AAC18871)
Putative serine-type carboxypeptidase II from A. thaliana
(AAL33815)
Ribulose-5-phosphate-3-epimerase from O. sativa (XP_470294)
Aldehyde dehydrogenase mRNA from A. thaliana (NM_179476)
Pathogenesis-related protein, putative from A. thaliana
(NP_195098.1)
Pathogenesis-related family protein from A. thaliana
(NP_849901.1)
Ribosomal protein L35 from A. thaliana (CAA60774.1)
60S ribosomal protein-like mRNA from A. thaliana (AY093180.1)
Putative ribosomal protein S3a homologue from A. thaliana
(AAL15196)
Putative 60S ribosomal protein from A. thaliana (AAN31827)
Putative 60S ribosomal protein L6 from A. thaliana (AAO00948)
Putative ribosomal protein S9 from A. thaliana (AAG51916)
40S ribosomal protein S3 mRNA from A. thaliana (NM_115247)

3e-68

24 h, 6 weeks

24 h, 6 weeks

24 h, 6 weeks
0

7.2e-10*

24 h

8e-13
5e-10
2e-06*
2e-07

0
24 h, 6 weeks
24 h
0

1e-15*
6e-07

24 h
6 h, 24 h, 6 weeks

3e-29

24 h, 6 weeks

6e-05
4e-51

6h
24 h, 6 weeks

2e-35
1e-48
3e-51

6h
6h
6h

3e-25

6h

1e-21
2e-06

6 weeks
6 weeks

5e-10
2e-06*
2e-15

0
24 h
6h

2e-53

6h

6e-07
7e-11*
9e-05

0
24 h
6h

8e-17
8e-12
2e-08
4e-06*

0
0
6 h, 24 h, 6 weeks
0

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2e-65

24 h, 6 weeks
24 h, 6 weeks

Cadmium-modulated genes in B. juncea

3021

Table 1. (Continued)
Length
(bp)

Accession
number

Homologya

BLAST
scoreb,c

Expression
patternd

BjCdR49

149

DT317704

3e-23

24 h

BjCdR50
BjCdR51

262
512

DT317705
DT317706

2e-16*
5e-93

6h
24 h

BjCdR52
BjCdR53

148
386

DT317707
DT317708

9e-08
2e-55

6h
6h

BjCdR54

134

DT317709

7e-05*

6 weeks

BjCdR55

147

DT317710

3e-16*

6h

BjCdR56

404

DT317711

2e-56

0, 6 h

BjCdR57

396

DT317712

8e-55

6 h, 24 h

BjCdR58

150

DT317713

1e-06*

6h

BjCdR59

149

DT317714

9e-08

6h

BjCdR60

96

DT317715

7e-11

BjCdR61
BjCdR62

128
250

DT317716
DT317728

8e-36*
3.3e-17*

6 h, 24 h
6h

BjCdR63

280

DT317717

0.00054*

6h

BjCdR64
BjCdR65

260
171

DT317718
DT317719

BjCdR66
BjCdR67
BjCdR68
BjCdR69
BjCdR70
BjCdR71
BjCdR72
BjCdR73

170
222
203
138
103
188
256
339

DT317720
DT317721
DT317722
DT317723
DT317724
DT317725
DT317726
DT317727

Plasma membrane intrinsic protein 2 (PIP2) from B. napus

(AAD39374.1)
SNF7 family protein mRNA from A. thaliana (NM_127541)
Plasma membrane intrinsic protein 1 (PIP1) from B. napus
(AAD39373.1)
Sec61beta family protein from A. thaliana (NP_182033.1)
Glutathione S-transferase, putative from A. thaliana
(NP_177957.1)
ERD9 mRNA for glutathione S-transferase from A. thaliana
(AB039930.1)
RNA-binding protein homologue (grp2A) mRNA from S. alba
(SALGRP2A)
Zinc finger (C3HC4-type RING finger) family protein from A.
thaliana (NP_194556.1)
DNAJ heat-shock N-terminal domain-containing protein from A.
thaliana (NP_178207.1)
Fasciclin-like arabinogalactan-protein, putative mRNA from A.
thaliana (NM_123780.2)
Tetratricopeptide repeat (TRP)-containing protein from A. thaliana
(NP_190782.3)
Radical SAM domain-containing protein/ TRAM
domain-containing protein from A. thaliana (NP_565035.1)
Probable protein kinase mRNA from A. thaliana (NM_104060)
Putative auxin-responsive protein mRNA from A. thaliana
(AK117242)
DNA chromosome 4, BAC clone F25I24 from A. thaliana
(ATF25I24)
Chromosome 2 clone from A. thaliana (AC004697)
Chromosome 2 clone F26B6 map CIC06C07 A. thaliana
(AC003040)
No homologye
No homology
No homology
No homology
No homology
No homology
No homology
No homology

9e-06*
1.1e-09*

6 weeks
24 h
6
6
24
24
6
6
6
0

h
h, 24 h, 6 weeks
h, 6 weeks
h, 6 weeks
weeks
h
h

GenBank accession numbers of sequences homologous to AFLP fragments are in parentheses.

All are BLASTX scores except for those marked with * which are BLASTN scores.
c
e-value cut-off=1e5.
d
Gene expression pattern after exposure to 10 lM Cd(NO3)2 for different times. 0, 6 h, 24 h, and 6 weeks (6 w) represent the Cd exposure time.
e
No significant sequence homology found in genome, EST, and protein database.
b

region of the transcripts, which is usually less conserved.

For three (4.1%) TDFs, sequences match only genomic
clones without allocated function.
Considering the expression pattern of Cd-modulated
genes, among the TDFs isolated and sequenced, Table 1
shows that genes were either up- or down-regulated by Cd
treatment. It is possible to distinguish 14 genes that were
expressed only in untreated control plants and downregulated by Cd treatment, while seven genes were induced
after Cd exposure and the transcription was sustained
throughout the Cd treatment (6 h, 24 h, and 6 weeks).
Table 1 also shows that 27 genes, the majority of the TDFs
identified, are detected within 6 h of exposure to Cd and
their expression is suppressed with longer Cd treatment
times, suggesting that many of them may have roles in
further signalling. Ten genes were up-regulated only 24 h
after the addition of Cd and three genes were induced

at 6 h and 24 h of Cd treatment. For six genes the transcription was observed at 24 h and 6 weeks of Cd exposure, and
only one was up-regulated in untreated plants and after
the addition of Cd for 6 h. In plants treated with Cd for
6 weeks the transcript accumulation of five genes was detected. The induction of the latter genes might probably
be due to general stress conditions imposed on the plants
by the continuous presence of Cd in the culture medium.
Indeed, protracted exposure to Cd inhibited growth, and
the generally stunted conditions and leaf chlorosis indicated
that general metabolism such as photosynthesis and respiration were affected.
Northern analysis of Cd-regulated genes

To validate the cDNA-AFLP expression patterns, several

TDFs were selected for RNA gel blot analysis. To minimize
problems of cross-hybridization, full-length TDFs were

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TDF

3022 Fusco et al.

used and their kinetics of transcript accumulation in response to the Cd presence in the culture medium are shown
in Fig. 3. Moreover, to confirm the expression pattern
observed, each hybridization was repeated at least twice
(data not shown). For this expression analysis, TDFs from
genes induced at 6 h of Cd treatment were preferentially
chosen as probes to further characterize genes which may
have roles in the early events of the signal transduction
pathways leading to Cd translocation and sequestration in
B. juncea.
The induction pattern observed in northern analysis
showed that six of the nine TDFs tested (BjCdR15,
BjCdR7, BjCdR35, BjCdR14, BjCdR29, and BjCdR30)

Functional classication and temporal expression

pattern of Cd-responsive genes

Fig. 3. Northern analysis showing the expression profiles of several

genes induced by Cd treatment in B. juncea. These experiments were
repeated at least twice for each TDF. Poly (A)+ RNA was isolated from
whole plants after Cd exposure for 0, 6 h, 24 h, and 6 weeks.

Of the 73 sequenced Cd-modulated genes, 52 were genes of

known or putative function which could be grouped into
several major functional categories (Table 2). Four were
transcriptional factors possibly involved in the transcriptional control of plant stress response. BjCdR15 and
BjCdR14 activated at 6 h of Cd treatment showed homology to Arabidopsis TGA3 and GBF5, respectively, which
belong to the bZIP protein family of transcriptional factors
that have been implicated in stress signalling (Jakoby et al.,
2002). In particular, the gene corresponding to BjCdR15 is
under investigation and preliminary data suggest that it is
transcribed in leaves and roots after 0.5 h of Cd treatment
and is also activated by other heavy metals, such as Pb and
Zn (Micheletto et al., unpublished results). In addition,
a cDNA fragment (BjCdR12) homologous to the Arabidopsis Myb59 was induced at 6 h of Cd treatment, while
BjCdR16 expressed at 6 and 24 h of Cd exposure showed
homology to zinc finger protein. Cd modulated the expression of several stress-responding proteins. Genes whose
expression improves plant stress protection were considered to be in this functional category. At 24 h of Cd stress,
BjCdR39 was isolated, which showed homology to an
aldehyde dehydrogenase induced by dehydration, NaCl,
heavy metal, and oxidative stress (Sunkar et al., 2003),
whereas 6 h of Cd stress induced the transcription of a gene
(BjCdR55) homologous to Sinapis alba mRNA cap-binding protein. It has been reported that the Arabidopsis
homologue is implicated in abscisic acid (ABA) signalling
(Hugouvieux et al., 2001). Two cDNA fragments
(BjCdR40 and BjCdR41) induced early by Cd exhibited
homology to pathogenesis-related protein. The stressresponding category also includes a gene (BjCdR24)

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fully confirmed the expression profiles observed with the

cDNA-AFLP analysis. This technique was thus validated in
67% of cases. It is noteworthy that there are slight differences in expression pattern for the three TDFs (BjCdR47,
BjCdR43, and BjCdR51) for which the transcription profile
seen with the cDNA-AFLP analysis was not confirmed by
RNA gel blot analysis. Northern analysis showed that the
ribosomal protein S9 (BjCdR47) is mainly induced at 6 h
and 24 h of Cd exposure and that its transcription decreased
with longer Cd treatment; whereas the 60S ribosomal
protein (BjCdR43) showed the highest transcript level
when Cd was added to the culture medium for 6 h, and
the plasma membrane intrinsic protein 1 (BjCdR51) is
mainly expressed at 24 h and 6 weeks of Cd treatment.
These discrepancies between northern blotting and cDNAAFLP analysis may either be due to changes in the intensity
of individual bands in the cDNA-AFLP gels or to gene
family complexity. Nevertheless, the cDNA-AFLP technique allowed the isolation of differentially expressed
genes under the conditions tested.

Cadmium-modulated genes in B. juncea

3023

Table 2. Functional classification of Cd-responsive gene product in B. juncea

Functiona

Identification of BjCdR clones

No. clones

Transcriptional factor
Stress responding
Cellular metabolism and organization
Photosynthetic process
Transport facilitation
Unclassified protein
Expression regulator
Miscellaneous
No hit b
Total

12, 14, 15, 16

40, 41, 53, 54, 57, 36, 22,
26, 27, 29, 20, 34, 37, 58,
17, 18, 19, 32, 21, 23, 25
33, 52, 50, 51, 49, 11
1, 2, 3, 4, 5, 6, 7, 8, 9, 10
42, 43, 44, 45, 46, 47, 48,
35, 61, 62, 60
63, 64, 65, 66, 67, 68, 69,

4
12
8
7
6
10
11
4
11
73

5.5
16.4
11.0
9.6
8.2
13.7
15.0
5.5
15.1
100

24, 39, 55, 38, 28

59

30, 31, 56, 13

70, 71, 72, 73

Unclassified protein indicates sequence that is homologous to unknown, putative, and expressed proteins without annotated function in other
organisms. Other sequence homologies denoted in Table 1 with a putative or probable function are included in their probable function categories.
b
No hit indicates identity only to unannotated genomic sequences or low similarity to existing nucleotide sequences.

The presence of Cd in plants causes an overall inhibition

of photosynthesis. In this work, seven genes related to the
photosynthetic process were found to be modulated by Cd.
The expression of three of them (BjCdR18, BjCdR21, and
BjCdR23) was inhibited by the presence of Cd in the
culture medium, while three cDNA fragments (BjCdR17,
BjCdR19, and BjCdR32) were detected only after 6 h of Cd
exposure, and BjCdR25 was found instead at 24 h of Cd
treatment. The presence of Cd in the culture medium
rapidly induced the synthesis of six proteins that were
included in the transport facilitation category. Following
the expression pattern in Table 1, BjCdR49 and BjCdR51
were isolated at 24 h of Cd treatment, which showed
sequence similarity to Brassica napus aquaporins PIP2 and
PIP1, respectively, although northern analysis (Fig. 3)
indicated that PIP1 is also transcribed at 6 weeks of Cd
exposure. The expression of these genes suggests that Cd
evoked water stress. After 24 h of Cd addition, BjCdR11
homologous to an Arabidopsis ABC1 transporter was also
induced. Moreover, genes with similarity to an AAA-type
ATPase (BjCdR33), Sec61 beta (BjCdR52) and SNF7
(BjCdR50), all involved in protein transport across membranes, were only induced at 6 h of Cd treatment.
Genes encoding for ribosomal proteins (BjCdR42,
BjCdR43, BjCdR44, BjCdR45, BjCdR46, BjCdR47, and
BjCdR48) were classified as expression regulators. The
transcription of four of them (BjCdR42, BjCdR45, BjCdR46,
and BjCdR48) was only observed in untreated control
plants. BjCdR47 was up-regulated by Cd and its transcription was maintained at all times of Cd treatment, although
northern analysis (Fig. 3) showed the higher expression at 6 and 24 h of Cd treatment. The expressions of
BjCdR44 and BjCdR43 were induced at 6 h and 24 h of
Cd exposure, respectively. Also for the latter gene, an RNA
gel blot (Fig. 3) revealed the highest expression after 6 h of
Cd addition. BjCdR30, homologous to a histone deacetylase, up-regulated at 6 h of Cd treatment, BjCdR31 with
similarity to a ubiquitin carrier protein, activated at 24 h
and 6 weeks of Cd treatment and probably involved in
protein degradation, were also included in this functional

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transcribed at 24 h and 6 weeks of Cd treatment that

showed homology to a gene encoding for an early lightinduced protein (ELIP) from Brassica rapa, and a gene transcribed at 24 h of Cd stress (BjCdR22) homologous to the
Arabidopsis cytochrome P450. Transcript induction was
observed in all Cd time treatments for a gene encoding Oacetylserine (thiol) lyase (BjCdR28), an enzyme that plays
a role in Cd tolerance (Domnguez-Solis et al., 2001).
Furthermore, BjCdR53 and BjCdR54, detected, respectively, at 6 h and 6 weeks of Cd exposure, showed high
homology to Arabidopsis genes encoding glutathione Stransferase directly involved in Cd stress tolerance. Not
unexpectedly, at 6 h and 24 h of Cd exposure, a cDNA
fragment (BjCdR57) was isolated homologous to a gene
encoding for a DNAJ heat-shock protein probably participating in protein folding, and BjCdR36 detected after 6
weeks of Cd treatment showed homology to an Arabidopsis
gene encoding for a phenylalanine ammonia lyase, a key
enzyme leading to lignin synthesis (Mao et al., 2004).
A group of eight genes were considered to be related to
cellular metabolism and organization. Fragments homologous to Arabidopsis chorismate mutase (BjCdR20), and
b-hydroxyacyl-ACP (BjCdR29) were observed at 24 h
and 6 weeks of Cd treatment and are involved in amino acid
and fatty acid biosynthesis, respectively, while BjCdR34
observed at 6 h of Cd exposure showed similarity to a gene
encoding a NADH-ubiquinone oxidoreductase involved in
cellular respiration. BjCdR26 and BjCdR27, which are
down-regulated by Cd or induced at 24 h of Cd treatment,
respectively, showed homology to genes encoding glutamine-synthetase, an enzyme involved in the synthesis of
glutamate. In addition, cDNA fragments detected after long
Cd exposure (BjCdR37) showed similarity to a serine
carboxypeptidase probably involved in plant development,
whereas BjCdR59 and BjCdR58 detected at 6 h of Cd
treatment were similar to a gene encoding a tetratricopeptide
repeat-containing protein implicated in proteinprotein interaction (Das et al., 1998) and a fasciclin-like arabinogalactan-protein involved in cell adhesion (Johnson et al.,
2003), respectively.

3024 Fusco et al.

category. Furthermore, transcription was inhibited by Cd

for a gene homologous to an initiation factor BjCdR13,
whereas inhibition was seen after 24 h and 6 weeks of
Cd exposure for a gene similar to a zinc finger RING
(BjCdR56).
The other four clones were clustered as miscellaneous. A
PBS lyase HEAT-like repeat-containing protein, BjCdR35,
was detected at 6 h of Cd treatment; a putative protein
kinase, BjCdR61, probably involved in signalling is activated at 6 h and 24 h of Cd treatment and a putative auxinresponsive protein, BjCdR62, is transcribed at 6 h of Cd
exposure. A gene encoding for a protein containing the
SAM and TRAM domain, BjCdR60, repressed by Cd is
also included in this functional category.
Discussion

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Some plants species can grow on soil that naturally, or due

to human activities, contains growth-prohibiting concentrations of metals (Clemens, 2001). For instance, Brassica
juncea, a fast-growing plant with high biomass, has the
ability to take up Cd, as other heavy metals, from the soil
and to accumulate substantial amounts of it in the shoot
(Salt et al., 1995a, 1997). Therefore, B. juncea is an
interesting species to use in the study of Cd accumulation
and tolerance mechanisms (Clemens et al., 2002; Rugh,
2004). In this study, cDNA-AFLP technology was
employed to identify Cd-regulated gene expression in B.
juncea to gain new insights into the molecular mechanisms
governing Cd accumulation.
Plants of B. juncea grown in hydroponic culture with Cd
added for 6 weeks showed symptoms of toxicity. Long
exposure to Cd induced leaf chlorosis. This observation is
in agreement with other works describing a reduction of
chlorophyll concentration in plants of B. juncea (Salt et al.,
1995b) and B. napus (Larsson et al., 1998) exposed to Cd,
indicating a general inhibition of the photosynthetic process. Cd also inhibited plant growth, affecting roots to
a greater extent than leaves, while plants exposed to Cd for
short times (6 h and 24 h) showed no symptoms. Similar
root growth inhibition was described for seedlings of
several genotypes of Pisum sativum grown in hydroponic
culture for 10 d and exposed to 5 lM CdCl2 (Metwally
et al., 2005). Long exposure to Cd affected root morphology; not only root elongation was inhibited, but roots
appeared thinner and sent out side roots. A plant with
numerous thin roots would accumulate more metals than
one with a few thick roots (Schierup and Larsen, 1981),
plus, to take up heavy metal ions, plants must be able to
renew the active parts of their root biomass (Das et al.,
1997). The development of thin and side roots in plants
exposed to Cd during growth may thus represent an
adaptive strategy to cope with Cd ions.
As a first step in the identification of Cd-modulated genes
in B. juncea, Cd contents of whole plants were measured

and similar plant materials were used for the cDNA-AFLP

analysis. The Cd content analysis showed that B. juncea
plants grown in hydroponic solution take up Cd and the
metal concentration in plants increased with the duration of
Cd treatment. The cDNA-AFLP technique allowed transcription changes to be surveyed with no prior assumptions
about which genes might be induced or repressed by Cd
treatment. Northern blotting analysis of nine TDFs confirmed that expression of the identified genes is Cd modulated. With the enzyme combination chosen, 3000 cDNA
fragments were visualized on the gels, and about 100 were
found to be Cd regulated, of which 73 are reported in
Table 1. They belong to different functional categories,
which indicates that Cd affected different physiological
and biochemical pathways. Sequencing analyses revealed
that one gene identified is homologous to a protein kinase (BjCdR61) and four encode transcriptional factors
(BjCdR12, BjCdR14, BjCdR15, and BjCdR16), indicating
that signal transduction pathways are rapidly activated by
the presence of Cd in the nutrient solution. Overall the
transcriptional factors identified in this work have been
linked to plant stress responses (Singh et al., 2002),
particularly TGA and GBF; members of the bZIP protein
family are involved in responses to ABA and ethylene as
well as in pathogen attack (Jakoby et al., 2002). The
induction by Cd of transcripts for bZIP, Myb, and zinc
finger transcriptional factor observed in this study suggest,
as previously reported by Knight and Knight (2001), that
plant response to environmental stresses, including heavy
metals, may be regulated by multiple signalling pathways.
Among the Cd-regulated genes detected, 12 appear to
encode proteins which protect against Cd stress. The
transcription upon exposure to Cd of a DNAJ heat shock
protein (BjCdR57), a chaperone involved in protein protection in times of cellular stress, confirmed that protein
denaturation is one of the effects of Cd toxicity (Suzuki et al.,
2001). Furthermore, the expression of two pathogenesisrelated proteins (BjCdR40 and BjCdR41) at 6 h after
Cd addition indicates that Cd induces defence reactions.
Indeed, H2O2 accumulation was observed in Cd-exposed
roots (Schutzendubel et al., 2001), and it was suggested
that H2O2 would act as a signalling molecule triggering
secondary defences leading to cell wall rigidification and
lignification in Cd-exposed cells (Schutzendubel and Polle,
2002). In addition, the transcript of phenylalanine ammonia lyase (BjCdR36), an enzyme of the phenylpropanoid
pathway leading to lignin synthesis, detected in 6-week-old
Cd-treated plants, is in accordance with the finding that
Cd induces lignin deposition in cell walls. One of the most
sensitive responses of higher plants to Cd is stomata
closure (Sanita` di Toppi and Gabbrielli, 1999), which is
a symptom of water stress mediated by ABA. Based on
transcript accumulation, aldehyde dehydrogenase has been
correlated with ABA treatment, drought stress, UV light,
NaCl, and heavy metals (Chen et al., 2002; Sunkar et al.,

Cadmium-modulated genes in B. juncea

(BjCdR19, BjCdR32, and BjCdR25) were induced by Cd

treatments, indicating that the presence of Cd in plant
tissues disturbs photosynthesis. Similarly, the effect of Cd
was evident from the expression pattern of genes that were
grouped under the function of cellular metabolism and
organization and that may be involved in different cellular
processes, such as a tetratricopeptide repeat-containing
protein (BjCdR59) induced after 6 h of Cd addition,
probably acting as a scaffold for the assembly of multiprotein complexes (Das et al., 1998), and a fasciclin-like
arabinogalactan-protein (BjCdR58) putatively working in
cell adhesion (Johnson et al., 2003). Furthermore, Cd
affected the transcription of genes encoding for glutaminesynthetase (GS) (BjCdR26 and BjCdR27) involved in
nitrogen metabolism. The expression of chloroplastic GS
was inhibited by Cd treatment, while transcription of the
cytosolic GS was detected 24 h after Cd addition. Protein
and transcript analyses of Cd-treated tomato plants showed
that cytosolic GS increased in leaves and chloroplastic GS
decreased in parallel, suggesting that plants subjected to Cd
stress induced cytosolic GS to compensate and continue
glutamine biosynthesis when Cd affected chloroplastic GS
activity (Chaffei et al., 2004). In addition, the induction
of chorismate mutase (BjCdR20) observed in this work at
24 h and 6 weeks of Cd exposure also confirms an increased amino acid synthesis in Cd-treated plants (Chaffei
et al., 2004).
Sequence analysis of Cd-responsive genes also revealed
the induction of genes whose gene products are involved in
cellular transport. Aquaporins mediate the passive movement of water in cellular membranes and the expression of
plasma membrane intrinsic proteins, PIP1 and PIP2, is
regulated by ABA and water stress (Gao et al., 1999; Yang
et al., 2003). The transcription of aquaporins PIP1 and PIP2
(BjCdR51 and BjCdR49) seen in B. juncea upon exposure
to Cd for 24 h, together with the expression of other droughtand ABA-responsive genes (BjCdR39 and BjCdR55)
strengthen the idea that Cd imposes water stress and
that both ABA and Cd act synergistically (Polle and
Schulzendubel, 2004). In addition, a gene encoding for an
ABC-transporter protein (BjCdR11) was up-regulated by
Cd treatment. ABC transporters directly involved in Cd
transport have not been identified in plants, whereas it has
been shown that in yeast and fission yeast they act directly
in the final step of Cd detoxification by mediating the
vacuolar transport of Cd complexes (Ortiz et al., 1995; Li
et al., 1997). However, recent analyses of AtMRPs, a
subfamily of Arabidopsis ABC transporters, showed that
AtMRP3 was induced by Cd and not by oxidative stress
(Bovet et al., 2003), suggesting that ABC transporters in
plants, as in yeast, are involved in heavy metal fluxes,
although a direct role of AtMRP3 in Cd transport has not
been demonstrated (Bovet et al., 2003).
In conclusion, the cDNA-AFLP technique allowed genes
to be identified whose expression is modulated by Cd. This

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2003). The expression of an aldehyde dehydrogenase

(BjCdR39) and an RNA-binding protein (BjCdR55)
involved in ABA signalling, induced by Cd in B. juncea,
corroborates the idea of existing cross-talk between Cdinduced and water stress-induced signalling that may also
employ ABA a as signal transduction compound (Polle and
Schutzendubel, 2004). Genes encoding for glutathione
S-transferases (BjCdR53 and BjCdR54) and cytochrome
P450 (BjCdR22), were previously reported as responsive to
Cd and other stresses (Marrs and Walbot, 1997; Suzuki
et al., 2001) and are probably functioning in cytotoxic product detoxification. In particular, glutathione S-transferases
catalyse the synthesis of glutathione S-conjugates, allowing
them to be recognized for transport into the vacuole (Marrs
and Walbot, 1997). Chelation of metals by high-affinity
ligands, such as PSs and thiolated peptides, is considered
a principal mechanism of Cd detoxification in plants, and
numerous physiological, biochemical, and genetic studies
have confirmed that glutathione is the substrate for PS
biosynthesis (Cobbett, 2000; Cobbett and Goldsbrough,
2002). Moreover, in B. juncea, changes of expression of
a glutathione transporter in response to Cd exposure has
been reported (Bogs et al., 2003) also indicating that
glutathione plays a prominent role in Cd accumulation
and/or detoxification. The amount of glutathione transporter protein decreased in older leaves 48 h after the onset
of Cd exposure and reached a minimum at 96 h, while an
increase in protein amount was detected at 120 h and 144 h
of Cd treatment. It was suggested (Heiss et al., 2003) that,
during Cd exposure, glutathione export to the various sink
tissues is reduced to meet the high demand of glutathione
for PS synthesis during Cd accumulation and, therefore, the
observed decrease in glutathione transporter protein may
reflect this adaptation. In this cDNA-AFLP analysis TDFs
for glutathione transporters were not detected. This may
simply be due to the technique employed that allows
discrimination between the presence or absence of bands,
or to the experimental conditions used. In this study, a
gene (BjCdR28) encoding for O-acetylserine (thiol) lyase
enzyme (OASTL) was up-regulated in all Cd exposure
times. OASTL catalyses the last step of cysteine biosynthesis and Arabidopsis plants overexpressing OASTL
showed high Cd resistance, suggesting that cysteine pool
requirement for glutathione biosynthesis is a main factor
for tolerance (Domnguez-Solis et al., 2001). However,
previous studies in B. juncea, also indicated an enhanced
OASTL expression, although glutathione biosynthesis,
more than cysteine availability, was considered the limiting
step (Schafer et al., 1998).
Cd toxicity is generally associated with inhibition of
chlorophyll synthesis and damage to photosynthetic apparatus (Sanita` di Toppi and Gabbrielli, 1999). In this work,
a group of genes related to the photosynthetic process
showed expression only in Cd-untreated control plants
(BjCdR18, BjCdR21, and BjCdR23), while others

3025

3026 Fusco et al.

study reveals that a multitude of processes are implicated in

determining response to metal in plants and these processes
require the activation of different sets of genes. Identification of components of the signal transduction cascades
suggests that Cd triggers stress signals and that ABA may
be involved in plant response to Cd. Detailed characterization of several genes, including putative novel genes and
genes of unknown function, which may be involved in
specific processes, will help to unravel the fine networks
underlying heavy metal accumulation and tolerance in
plants.
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