Botanical Studies (2007) 48: 43-54.

3

The author made a contribution equal to the first one.

*

Corresponding author: E-mail:

zmyang@njau.edu.

c n;

Tel: +86-25-84395057, +86-25-84396248; Fax:

+86-25-84396673.

INTRODUCTION

Cadmium (Cd) is one of the most toxic of heavy

metals and is widely released into agricultural soils. Due

to its high mobility in soil, cadmium is readily taken up

and accumulated by plants. Excess Cd

2+

accumulation

in plants leads to impaired growth and development and

even death (Sanita di Toppi and Gabbrielli, 1999). Several

mechanisms have been proposed for Cd toxicity in plants

(Clemens, 2001). Cd-induced generation of hydrogen

peroxide and other reactive oxygen species has been

correlated with the damage to plasma membrane lipids

and alteration of secondary metabolism (Schutzendubel et

al., 2001; Shah et al., 2001; Kuo and Kao, 2004). Plasma

membrane H

+

-ATPase activity and other enzymes related

to nitrogen metabolism are also sensitive to cadmium

(Astolfi et al., 2004, 2005). In addition, cadmium has been

shown to influence the sulfate uptake and assimilation in

plants (Lee and Leustek, 1999; Heiss et al., 1999; Nocito

et al., 2002).

Although most of plant species can not thrive under

the excessive heavy metal environment, some other

species have evolved sophisticated strategies for tolerating

high levels of heavy metals. Plants possess a range

of cellular mechanisms for detoxicification of heavy

metals (Rauser, 1999; Clements, 2001). Cd-induced

synthesis of metal chelating compounds like glutathione

(GSH), phytochelatins (PCs), and other sulfide-

enriched compounds is considered one of the important

mechanisms allowing plants to tolerate higher levels of

Cd in cells (Zenk, 1996; Cobbett and Goldsbrough, 2002).

Cd-induced PCs alleviate Cd toxicity by formation of Cd-

PC complexes, known as low-molecular-weight (LMW)

Cd complexes (Speiser et al., 1992; Cobbett, 2000). The

Cd-PC complexes are formed in cytosol and actively

Coordinated expression of sulfate transporters and

its relation with sulfur metabolites in Brassica napus

exposed to cadmium

Xue Mei SUN

1

, Bo LU

2,3

, Si Qi HUANG

1

, Surya Kant MEHTA

1

, Lai Lang XU

1

, and Zhi Min

YANG

1,

*

1

Department of Biochemistry and Molecular Biology, College of Life Science, Nanjing Agricultural University, Nanjing,

210095, P.R. China

2

Department of Chemistry, College of Sciences, Nanjing Agricultural University, Nanjing, 210095, P.R. China

(Received March 7, 2006; Accepted June 5, 2006)

ABSTRACT.

Full-length cDNAs encoding a putative low-affinity sulfate transporter (LAST), designated

BnSultr2;2 (accession number DQ091257) were isolated from Brassica napus 72 h after sulfate deficiency.

BnSultr2;2 shows significant similarity at an amino acid level with the available LASTs from the dicoty-

ledonous Arabidopsis and Brassica juncea. It displays the typical twelve-membrane spanning domains of

other plant species¡¦ sulfate transporters. Examination of steady-state mRNA levels reveals that BnSultr2;2

transcripts were enhanced in leaves of sulfate-deficient plants. The leaf BnSultr2;2 expression was also up-

regulated under 20-120 £gM Cd exposure, but under the same conditions, the BnSultr2;2 expression in roots

was severely suppressed. To understand the relationship between sulfate uptake and Cd stress, we simulta-

neously isolated another cDNA encoding a known high-affinity sulfate transporter (BnSultr1;1, accession

number AJ416460) from roots. RT-PCR analysis demonstrates that BnSultr1;1 was expressed only in roots,

and its expression was upregulated by both sulfate-deficiency and Cd exposure. To link up the expression of

sulfate transporters to sulfur accumulation and assimilation, the concentrations of sulfate, sulfide, glutathione,

and thiol-containing compounds in plant tissues were measured. Elevating Cd concentrations in the culture

medium up to 40 £gM increased the accumulation of sulfate, sulfide, glutathione, and non-protein thiols in

roots. These results suggest that a coordinated regulation of sulfate transporters enables plants to tolerate Cd

stress via an efficient sulfate uptake and assimilation.

Keywords: Brassica napus; Cadmium; Sulfate transporter; Transcriptional expression.

BIOChemISTRy

44

Botanical Studies, Vol. 48, 2007

transported from cytosol to the vacuole, where more

complicated and stable Cd-PC complexes are formed

(Erika and Laszlo, 2002; Hrustioger, 2000). Substantial

evidence shows that Cd-induced PC synthesis and metal

detoxification mechanisms involve a variety of molecular

and biochemical processes (Meuwly et al., 1995; Keltjens

and van Beusichem, 1998; Clemens, 2001). The process

of metal-induced synthesis of PCs most often leads to a

short-term depletion of the glutathione pool, one of the

major sources of thiol groups responsible for biological

redox processes and a precursor for the synthesis of PCs

(Leustek and Saito, 1999; Cobbett, 2000; Wang et al.,

2004). However, recovery in the glutathione pool up to

the level of or above controls has been demonstrated after

a prolonged exposure of plants to Cd (Vogeli-Lange and

Wagner, 1996; Schutzendubel et al., 2001). Enzymes

involved in GSH synthesis such as £^-glutamylcysteine

synthetase (£^-ECS, EC 6.3.2.2) and GSH synthetase (EC

6.3.2.3) have been examined in plants under metal stress,

and the activity of £^-ECS was found to be enhanced

upon Cd exposure (Schafer et al., 1998; Cobbett, 2000).

Molecular responses to Cd exposure have also been

identified in plants, such as Brassica juncea (Zhu et

al., 1999), Arabidopsis (Xiang and Oliver, 1998), and

the other plant species in which Cd exposure induced a

coordinated transcriptional regulation of genes encoding

£^-ECS, GSH synthase, and PC synthase (Schafer et al.,

1998; Zhu et al., 1999; Vatamaniuk et al., 2000; Sun et al.,

2005).

Cd-induced GSH and PC synthesis have a well es-

tablished relationship with sulfate uptake, transport, and

assimilation in plants (Lee and Leustek, 1999; Nocito

et al., 2002; Astolfi et al., 2004). For example, under Cd

stress a large amount of sulfate accumulates and the level

of thiol-containing compounds surges (Leustek and Saito,

1999; Nocito et al., 2002). Plants have a specific group of

root carriers for uptake of sulfate from soil (Leustek and

Saito, 1999; Hawkesford, 2003; Mendoza-Cozatl et al.,

2005). Equally, translocation of sulfate or thiol-containing

metabolites between the root and shoot is performed by

an array of sophisticated transport systems (Hawkesford,

2000; Howarth et al., 2003). In higher plants, sulfate

transporters are subdivided into five groups. Group 1

sulfate transporters have a high affinity for sulfate, and

normally facilitate primary uptake of sulfate from soils

into roots. AtSultr1;1, AtSultr1;2, and AtSultr1;3, isolated

from Arabidopsis (Vidmar et al., 2000), HVST1 from

barley (Smith et al., 1997) and SHST1, SHST2 from

Stylosanthes hamata (Smith et al., 1995) are typical high

affinity sulfate transporters, and they express mainly in

roots. Group 2 members such as AtSultr2;1 (AST56) and

AtSultr2;2 (AST68) from Arabidopsis and SHST3 from S.

hamata have a low affinity to sulfate and are expressed in

both roots and shoots (Smith et al., 1995; Takahishi et al.,

1996; Takahishi et al., 1997; Takahishi et al., 2000). Sug-

gestions have been made that this group of transporters

are principally involved in translocation, distribution

or reallocation of sulfate in plants (Smith et al., 1995;

Takahashi et al., 1997). Group 3 sulfate transporters are

localized in root, stem, leaf and even in root nodules of

Lotus japonicus (Takahashi et al., 1999; Buchner et al.,

2004a; Krusell et al., 2005), and the group 4 transporters

are positioned in tonoplast, suggesting that they facilitate

the efflux of sulfate from vacuoles (Kataoka et al., 2004).

Compared to Groups 1-4, the sequence divergence of

group 5 suggests that these may be functionally distinct

transporters. So far, genes encoding sulfate transporters

from a variety of higher plants have been identified and a

majority of these transporters are up- or down-regulated

at the transcriptional level under sulfate starvation

and restored (Hawkesford, 2003). However, a fine and

coordinated expression pattern of these genes in response

to heavy metal (e.g. Cd) stress has not been described.

Brassica napus falls into the same family as Brassica

juncea, which as a heavy metal accumulator has the poten-

tial to remove excessive heavy metals from contaminated

soils (Banuelos and Meek, 1990; Salt et al., 1995). Some

genotypes of B. napus exhibit a comparable or even

stronger tendency to accumulate heavy metals (Ebbs

and Kochian, 1997; Su and Huang, 2002). Since B.

napus produces as much high biomass as B. juncea, it is,

therefore, suitable for use in phytoremediation. The use

of higher plants in the phytoremediation of heavy metal-

contaminated soils is not only based on their ability to take

up and accumulate the metals, but also on mechanisms

able to alleviate their toxic effect (Salt et al., 1998; Nocito

et al., 2002). However, little is known about the process of

Cd tolerance in B. napus. Understanding of the molecular

and physiological regulatory mechanisms of plant sulfate

transporters may provide a better insight into our un-

derstanding of Cd tolerance in plants. The present study

examined the Cd-induced transcriptional expression of

BnSultr2;2 and BnSultr1;1 and its correlation with the

sulfur accumulation and assimilation in B. napus.

mATeRIALS AND meThODS

Plant material and treatment

Seeds of B. napus L. (cv. Youyan No. 9) were sterilized

with 5% NaClO for 10 min, washed several times with

distilled water, and germinated for 3 d in the dark on float-

ing plastic net. After germination, nine young seedlings

were transferred to 1 L polyethylene containers containing

1/4-strength modified Hoagland nutrient solution (0.7 mM

Ca

2+

, 1.5 mM K

+

, 0.5 mM

Mg

2+

, 0.25 mM NH

4

+

, 2.9 mM

NO

3

-

, 0.25 mM

H

2

PO

4

-

, 0.5 mM

SO

4

2-

, 4.75 £gM Fe

2+

, 0.32

£gM Cu

2+

, 0.2 £gM Zn

2+

, 1.25 £gM Mn

2+

, 11.5 £gM H

3

BO

3

,

0.025 £gM MoO

3

). Seedlings were grown at 22¡Ó1oC, with a

light intensity of 200 £gmol

m

-2

s

-1

and a 14-h photoperiod.

After growing for 14 days, plants were treated with 0, 20,

40, 80, and 120 £gM Cd as CdCl

2

in the 1/4-strength nutri-

ent solution as indicated above for 72 h, or with 0 and 40

£gM Cd for 0, 6, 12, 24, 48, 72 and 96 h. In the experiment

of sulfate deficiency, a sulfur-free solution was prepared

using MgCl

2

instead of MgSO

4

. The pH of culture as well

SUN et al. ¡X Sulfate transporters in response to Cd

45

as of treatment solutions was adjusted to 5.6. Treatment

solutions were renewed with freshly prepared nutrient so-

lution each day. After treatment, root and leaf tissues were

separately harvested and immediately frozen in liquid ni-

trogen and stored at -80¢XC in a freezer.

RNA isolation and cDNA synthesis

Total RNA was extracted from root or leaf tissues using

Trizol (Invitrogen, Carlsbad, CA) according to the manu-

facturer ¡¦s instructions. Reverse transcription was carried

out at 37oC in 25 £gL reaction mixture containing 3 £gg

of RNA, 0.5 £gg oligo (dT) primer, 12.5 nmol dNTPs, 25

units of RNase inhibitor and 200 units of M-MLV reverse

transcriptase (Promega, Madison, WI). The first strand

cDNA was then used as a template for polymerase chain

amplification.

Cloning of a putative low-affinity sulfate

transporter

The amplification primers for partial BnSultr2;2 in B.

napus were 5¡¦-AAC CCT AAT CGT GTA TTT-3¡¦ (sense)

and 5¡¦-GGA CAA TAT ATC ATT TAT TCT G-3¡¦ (anti-

sense) based on Indian mustard (Brassica juncea) partial

low affinity sulfate transporter mRNA (accession number

AJ223495). The amplification product was cloned into the

vector pGEM-T (Promega, Madison, WI) and sequenced

(SEQLAB, Gottingen, Germany). The resulting partial

cDNA sequence containing 3

¡¬

UTR showed a 93% identity

with Brassica juncea and an 86% identity with Arabidop-

sis thaliana putative sulfate transporter mRNA (accession

number AY074516). Another pair of primers was designed

to amplify 5¡¦-region to obtain the full-length cDNA. The

sense primer based on Arabidopsis thaliana was 5¡¦-CAC

TTC AAT AAC CCA CAA-3¡¦, and the antisense primer

based on partial BnSultr2;2 was 5¡¦-GCA AGA AGT TAA

AGA GCC-3¡¦. The amplified 1.2 kb fragment was cloned

into the vector pGEM-T and sequenced. To obtain 5¡¦ UTR,

the gene-specific primers for 5¡¦-RACE were: RACE-5A:

5¡¦-GCA AGA AGT TAA AGA GCC-3¡¦, RACE-5B: 5¡¦-

CGA AAC TAC AGC CAC AGG ACC A -3¡¦ and RACE

5C: 5¡¦-ACC G TA CTC TGG ATC AAG TC-3¡¦.

In order to create the full-length cDNA of BnSultr2;2

from B. napus by RT-PCR, a 2144 bp fragment was am-

plified with ExTaq DNA polymerase (Takara) and cloned

into the vector pGEM-T for sequencing.

Determination of BnSultr1;1 and BnSultr2;2

gene expression by RT-PCR

The first-strand cDNAs derived from the total RNA of

different treatments were used to analyze transcripts of

BnSultr1;1 (accession number AJ416460, Buchner et al.,

2004a) and BnSultr2;2 (accession number DQ091257,

this study). PCR was carried out using primers: Hast-S (5

¡¦-AGA TGA TCG CAT TGG GTA-3¡¦) and Hast-A (5¡¦-

CCT TTC TCG GAC ATA GTT G-3¡¦) for BnSultr1;1;

Last-S (5¡¦-TAC TTC ACA AAT TGA AAC GAG-3¡¦) and

Last-A (5¡¦-GGA CAA TAT ATC ATT TAT TCT G-3¡¦)

for BnSultr2;2. Expression of EF-1

£\

gene (sense 5¡¦-

AGACCACCAAGTACTACTGCAC-3¡¦ and antisense

5¡¦-CCACCAATCTTGTACACATCC-3¡¦) was used as a

control. RT-PCR products were obtained after 32 PCR

cycles. The PCR products were applied to 1% (w/v) aga-

rose gel electrophoresis and stained with ethidium bro-

mide. The strength of the fluorescent signal derived from

ethidium bromide in each lane was determined by the

software GIS Gel-ID produced by the Tanon Company.

Sequence analysis

Protein sequence homologies were calculated by the

DNAMAN program. Two protein alignments were per-

formed by the same program. The phylogenetic tree of

sulfate transporters was drawn using TreeView32 (Page,

1996) based on the multiple alignments performed by

Clustal X (Thompson et al., 1997). The transmembrane

analysis was carried out using TopRed (http://bioweb.pas-

teur.fr/seqanal/interfaces/toppred. html).

Determination of non-protein thiols and

glutathione

Non-protein thiols (NPT) were extracted by homog-

enizing 0.5 g plant material in 1 mL ice-cold 5% (w/v)

sulfosalicylic acid solution. After centrifugation at 10,000

g and 4¢XC for 30 min, the supernatants were collected and

immediately assayed for determination of NPT. NPT lev-

els in samples were measured with Ellman¡¦s reagent (Ell-

man, 1959). Briefly, 300 £gL of the supernatant was mixed

with 1.2 mL of 0.1 M phosphate buffer solution (pH 7.6).

After reaching a stable absorbance at 412 nm, 25 £gL of 5,

5¡¦-dithiobis (2-nitrobenzoic acid) (DTNB) solution (6 mM

DTNB dissolved in 5 mM EDTA and 0.1 M phosphate

buffer, pH 7.6) was added, and the increase in absorbance

at 412 nm was recorded.

Reduced glutathione (GSH) contents were determined

fluorimetrically (Hissin and Hilf, 1976). Plant material (0.5

g) was ground in 0.5 ml 25% H

3

PO

3

and 1.5 ml of 0.1 M

sodium phosphate-EDTA buffer (pH 8.0). The homogenate

was centrifuged at 10,000 g for 20 min. The supernatant

was further diluted 5 times with sodium phosphate-

EDTA buffer (pH 8.0). 100 £gL of diluted supernatant

was incubated with 1.8 ml of phosphate-EDTA buffer

and 100 £gL .-phthalaldehyde (1 mg ml

-1

) for 15 min at

room temperature to interact with the GSH in the solution.

Finally, the solution was transferred to quartz cuvette, and

the fluorescence at 420 nm was measured after excitation

at 350 nm.

Determination of total sulfate and sulfide

For determination of sulfate content in plant tissues,

plant samples were digested using HNO

3

:HClO

4

(8:1)

solution and turbidimetry was used to determine sulfate

according to method of Tababai and Bremner (1970).

Total sulfide in fresh plant tissues was assayed by the

methylene blue spectrophotometric method as described

by Huang and Wang (1997) with a few modifications.

46

Botanical Studies, Vol. 48, 2007

Plant samples were homogenized in an iced-cold 10 mM

NH

3

-NH

4

+

buffer (pH 10). The homogenate was centri-

fuged at 15,000 g at 4¢XC for 10 min. Supernatants from

the root sample were directly used to measure sulfide.

To avoid the interference of chlorophylls and pigments

with measurements, the supernatant from leaf tissues was

mixed with the CCl

4

:CHCl

3

(3:1) extraction solution to

remove these pigments. The proportion of the supernatant

to extraction solution was 1:1. The supernatant was used

for sulfide determination. Sulfide content was spectropho-

tometrically measured at 412 nm in a reaction mixture

containing 2 mL of 1% p-amino-N, N-dimethylaniline

(dissolved in 2 M H

2

SO

4

), 1 mL of 10 mM Fe

3+

and 1 mL

of supernatant.

Cadmium measurement

Root and leaf tissues were thoroughly rinsed with

deionized water and blotter dried. Samples were dried at

70

¢X

C in a forced-air oven, weighted, and digested with a

solution containing nitric acid and perchloric acid (1:1,

v/v). Cd in solution was determined by atomic absorption

spectrometry (Yang et al., 2001).

Statistical analysis

Each result shown in a table and figure was the mean

of at least three replicated treatments. Each treatment

included at least nine seedlings for determination. The

significance of differences between treatments was statisti-

cally evaluated by standard deviation and Student¡¦s t-test

methods.

ReSULTS

Cloning and analysis of a putative BnSultr2;2

from B. napus

The Arabidopsis genome contains at least 14 members

of sulfate transporter genes that are assumed to function

independently for the uptake and distribution of sulfate

in various cell types (Hawkesford, 2003). In Brassica

napus, five different sulfate transporter genes have

been cloned (accession number: AJ311389, AJ311388,

AJ581745, AJ416461, AJ416460). In this study, a

cDNA encoding a putative B. napus low-affinity sulfate

transporter, designated as BnSultr2;2, was cloned by

RT-PCR and 5¡¦ RACE. The BnSultr2;2 cDNA is 2150

bp long with an open reading frame (ORF) of 1959 bp,

a 35-nucleotide(nt)-5¡¦ untranslated region (UTR), and

a 153-nt 3¡¦ UTR. The ORF encodes a 653-amino acid

protein with a predicted molecular weight of 72 kDa and

an isoelectric point of 9.5.

The deduced protein includes twelve putative mem-

brane-spanning domains (Figure 1), consistent with the

general features of sulfate transporters (Smith et al.,

2000). The predicted protein was compared with a sulfate

transporter from A. thaliana (Figure 1) (Takahashi et al.,

1996) and several other higher plant sulfate transporters

(Table 1). The comparison shows a higher protein se-

quence similarity to low-affinity sulfate transporters than

to high-affinity sulfate transporters. To further analyze

the relationship between the B. napus sulfate transporter

and other higher plant sulfate transporters cloned so far,

a phylogenetic tree was constructed based on Clustal X

(Figure 2). The phylogenetic relationships among the plant

sulfate transporters indicated that the predicted protein of

BnSultr2;2 falls into the group 2 together with BjSultr2;2

from B. juncea (Heiss et al., 1999), BoSultr2.1 from B.

oleracea (Buchner et al., 2004a), AtSultr2;1 (Takahashi

et al., 1997) and AtSultr2;2 (Takahashi et al., 1996) from

A. thaliana, and SHST3 from Stylosanthes hamata (Smith

et al., 1995), respectively. This group of sulfate transport-

ers is clearly different from others, particularly the high-

affinity sulfate transporters of group 1, like SHST1 and

SHST2 in S. hamatus (Smith et al., 1995). Therefore, the

putative sulfate transporter BnSultr2;2 cloned from B. na-

pus should encode a low-affinity sulfate transporter.

Plant growth and Cd accumulation under the

Cd stress

Two-week-old plants of B. napus grown in the presence

of 10, 20, 40, 80 and 120 £gM Cd

2+

for 7 d showed a

concentration-dependent inhibition of growth (Figure

3A and 3B). A significant decline in root biomass was

observed at a 40 £gM external concentration of Cd, under

which the root dry weight decreased by 46% as compared

to the control. Shoot growth appeared to be more sensitive

to Cd as a relatively low concentration (20 £gM) of Cd lead

Table 1. Comparison of protein sequence similarity between BnSultr2;2 cDNA and other higher plant sulfate transporters.

Sulfate transporters accession number (species)

Sequence identity (%) with

BnSultr2;2 protein

Sulfate transporter (ST) type

AJ223495 (B. juncea)

93

Partial low-affinity ST (putative)

D85416 (A. thaliana, AtSultr2;2)

87

Low-affinity ST

AB003591 (A. thaliana, AtSultr2;1)

58

Low-affinity ST

X82454 (S. hamata, SHST3)

60

Low-affinity ST

X82455 (S. hamata, SHST1)

46

High-affinity ST

X82456 (S. hamata, SHST2)

46

High-affinity ST

SUN et al. ¡X Sulfate transporters in response to Cd

47

Figure 1. Alignment of the predicted amino acid sequences of BnSultr2;2 and AtSultr2;2. Nucleotide sequences are deposited with

GenBank under accession number DQ091257 (BnSultr2;2) and D85416 (Arabidopsis AtSultr2;2), respectively. The 12 membrane-

spanning domains (1-12) were identified by the boxes. The alignment was performed using the DNAMAN program.

Figure 2. Phylogenetic relationship of plant sulfate transporters. The phylogenetic tree was based on the alignment of protein

s equences of sulfate transporters using the Clustal X program. Accession number: Arabidopsis thaliana AtSultr1;1, AB018695;

AtSultr1;2, AB042322; AtSultr1;3, AB049624; AtSultr2;1, AB003591; AtSultr2;2, D85416; AtSultr3;1, D89631; AtS ultr3;2,

AB004060; AtSultr3;3, AB023423; AtSultr3;4, AB054645; AtSultr3;5, AB061739; AtSultr4;1, AB008782; AtSultr4;2, AB052775;

AtSultr5;1 NP_178147; AtSultr5;2, NP_180139; Stylosanthes hamata: SHST1, X82255; SHST2, X82256; SHST3, X82454; Brassica

oleracea: BoS ultr1;3, AJ633707; BoS ultr2;1, AJ633705; BoS ultr3;1, AJ581745 BoSultr3;2, AJ601439; BoSultr3;3, AJ704373;

BoSultr3;4, AJ704374; BoSultr3;5, AJ633706; BoSultr4;2, AJ555124; Brassica napus: BnSultr1;1, AJ416460; BnSultr1;2, AJ311388;

BnSultr2;2, DQ091257 (this study), BnSultr4;1, AJ416461; BnSultr5;1, AJ311389; Brassica juncea: BjSultr2;2, AJ223495.

48

Botanical Studies, Vol. 48, 2007

to a significant inhibition of leaf growth. Treatment with

Cd at 40 £gM decreased the shoot growth by 43%. Since

40 £gM Cd triggered a moderate effect on the plant growth,

this concentration was selected to examine transcriptional

and physiological responses of B. napus to Cd stress.

Figure 4 illustrates the time-course of Cd accumulation

in the root and shoot of seedlings from nutrient solution

containing 40 £gM Cd. Cd accumulation in roots began

immediately after exposure of seedlings to Cd. The

accumulation of Cd in root was rapid and nearly linear

during the first 12 h of exposure and then slowed down

up to experiment end. A maximum of 3634 mg kg

-1

Cd

accumulated in roots after a 96-h exposure of seedlings

to 40 £gM Cd. Meanwhile, less Cd accumulated in shoots

than in roots. The highest Cd level in shoots was 371.4 mg

kg

-1

.

effects of sulfur deficiency on the transcri-

ptional expression of sulfate transporters

To examine the response of BnSultr2;2 expression to

different sulfur conditions, 2-week-old plants grown on

the sulfur-sufficient medium, were transferred into the

sulfur-deficient solution for 3 days. Withdrawal of external

sulfate supply did not affect the level of root BnSultr2;2

mRNA (Figure 5A). However, significant BnSultr2;2

mRNA did accumulate in leaves under the sulfur deficien-

cy. The abundance of BnSultr2;2 mRNA in sulfur-deficient

leaves was 8.8 times higher than in sulfur-sufficient

leaves. To understand further the regulation of sulfate

uptake and accumulation, we simultaneously isolated

another cDNA encoding a known high-affinity sulfate

transporter BnSultr1;1 (accession number AJ416460) from

roots. In contrast to BnSultr2;2, BnSultr1;1 appeared to

be specifically expressed in roots, and its transcript level

there increased by 5.2 fold over the control (Figure 5C).

effects of Cd on the expression of BnSultr2;2

and BnSultr1;1

To examine whether BnSultr2;2 and BnSultr1;1

expressions were regulated by Cd concentration in nutri-

ent medium, 2-week-old plants were grown continuously

for 72 h in hydroponic culture containing 0, 20, 40, 80 and

120 £gM Cd. In roots, BnSultr2;2 transcript level declined

progressively with increasing concentration of Cd in

media (Figure 6A). At the 40 £gM external concentration

of Cd, the mRNA transcript level of BnSultr2;2 was only

12% of the control. The BnSultr2;2 transcript level in

leaves increased with increasing Cd concentration in the

nutrient solution, and at a 40 £gM external concentration

of Cd the highest increase in mRNA level was noted. Fur-

ther increase in Cd concentration resulted in a decreased

expression of BnSultr2;2 in leaves of B. napus (Figure

6B). The level of BnSultr1;1 transcripts in the root of Cd-

treated plants was higher than in the control, and the maxi-

mum accumulation of BnSultr1;1 transcripts was observed

at 80 £gM of Cd in nutrient solution (Figure 6C).

A time-course experiment was also performed to

investigate the transcriptional response of BnSultr2;2

and BnSultr1;1 to Cd exposure. The root BnSultr2;2

expression was inhibited during the time-course of Cd

exposure (Figure 7A). BnSultr2;2 expression in leaf

showed a different time-course pattern when com-

pared with root (Figure 7B). Only a small amount of

BnSultr2;2 transcript was detected during the first 12 h of

Cd treatment. A rapid induction in level of mRNA tran-

script level was found after 12 h of exposure to Cd. The

BnSultr2;2 expression in leaf peaked 48 h after Cd treat-

Figu re 3. Effect of Cd on growth of B. napus roots (A) and

shoots (B). Seedlings were incubated on the 1/4 strength of Hoa-

gland solutions containing the indicated concentrations of Cd

for 7 days, and then roots and shoots were harvested, dried, and

analyzed for biomass. Vertical bars represent standard deviation

of the mean (n=20). Asterisks indicate that mean values are sig-

nificantly different between the treatment and control (p<0.05).

Figure 4. Time-course of Cd accumulation in roots and shoots

of B. napus seedlings subjected to 40 £gM Cd. Vertical bars rep-

resent standard deviation of the mean (n=3). Asterisks indicate

that mean values are significantly different between the treat-

ment and control (p<0.05).

SUN et al. ¡X Sulfate transporters in response to Cd

49

ment. After peaking, however, it sharply decreased and

returned to the level of control. For BnSultr1;1, treatment

with 40 £gM Cd did not induce its expression during the

first 12 h, but a progressive increase in expression was

observed thereafter. The highest expression was reached at

72 h (Figure 7C).

effects of Cd on the accumulation and sulfate,

sulfide, and non-protein thiols

To relate expression of the sulfate transporters to

sulfur accumulation and assimilation, the content of

sulfate, sulfide, and non-protein thiols in plant tissues

were measured. Treatments of seedlings with Cd at

concentrations ranging from 0-120 £gM induced a consis-

tent enhancement in sulfate accumulation in roots (Figure

8A). A significant increase in sulfate content was evident

at 40 £gM. Time-course study demonstrated that roots

treated with 40 £gM Cd also exhibited an accumulation of

sulfate with the time, but significant increases occurred

only after 24 h of the treatment with Cd (Figure 8C). By

contrast, sulfate accumulation in leaves consistently de-

creased as evinced by dose-response (Figure 8B) as well

as time-course (Figure 8D) study.

Figure 5. Analysis of transcript amounts of BnSultr2;2 (A and B)

and BnSultr1;1 (C) in the tissues of B. napus under the sulfate

deprivation. Plants were grown hydroponically for 2 weeks

before transfer to sulfate-deficient medium for 72 h. Control

plants (+S) were maintained on sulfate-containing hydroponic

medium. RNA was extracted from root and leaf tissues. EF-1£\

was used for cDNA normalization.

Figure 6. Analysis of transcript amounts of BnSultr2;2 („S) in

roots (A) and leaves (B) and BnSultr1;1 („S) in roots (C) of B.

napus in response to Cd. Plants were grown hydroponically for

2 weeks and then transferred to the same solution containing Cd

at 0, 20, 40, 80, and 120 £gM for 72 h. RNA was extracted from

root and leaf tissues. EF-1£\ („T) was used for cDNA normaliza-

tion.

Figure 7. Analysis of transcript amounts of BnSultr2;2 („S) in

roots (A) and leaves (B) and BnSultr1;1 („S) in roots (C) of B.

napus in response to Cd. Plants were grown hydroponically for

2 weeks and then transferred to the same solution containing

40 £gM Cd for 0, 6, 12, 24, 48, 72 and 96 h. RNA was extracted

from root and leaf tiss ues . EF-1£\ („T ) was us ed for cDNA

normalization.

50

Botanical Studies, Vol. 48, 2007

Sulfide (S

2-

) is one of the important intermediates

in the reductive SO

4

2-

assimilation pathway, in which

the incorporation of sulfide into cysteine is the last step

(Leustek and Saito, 1999). Thus, the level of sulfide

in

plants may reflect their capacity for sulfate reduction

and assimilation. Our results show that elevating Cd

concentrations from 0 to 80 £gM in the culture medium

resulted in a massive accumulation (12-fold of control)

of sulfide in roots (Figure 9A). However, only a little

induction was observed in leaves. Because reduced

glutathione (GSH) is thought to play an important role in

heavy metal detoxification, the concentration of GSH in

Cd-treated plants was measured. Cd exposure (40 and 80

£gM) significantly increased the concentrations of GSH in

roots. Compared with roots, the content in leaves was not

obviously affected by Cd (Figure 9B).

The total non-protein thiols were measured because

these thiols represent the compounds of monothiols (e.g.

cysteine and glutathione) and polythiols (phytochelatin)

(Cobbett, 2000). As shown in Figure 10A, the presence

of 20 £gM Cd was sufficient to stimulate the production

of total non-protein thiols in roots. A concentration-

dependent change in thiol abundance was observed.

The production of total non-protein thiols in Cd-treated

roots also gradually elevated over the time (Figure 10C).

However, in the present study, Cd exposure did not induce

any leaf non-protein thiol accumulation (Figure 10B and

D).

DISCUSSION

In this study we isolated and partially identified a

novel cDNA (BnSultr2;2, accession number DQ091257)

encoding Brassica napus low-affinity sulfate transporter.

Sequence comparison of deduced protein of BnSultr2;2

with other sulfate transporters¡Xin particular those of

A. thaliana (Takahashi et al., 1996, 1997), S. hamata

(Smith et al., 1995), and B. juncea (Heiss et al., 1999)¡X

has shown that the cloned putative LAST should be

classified as a member of group-2 sulfate transporters. The

Figu re 8. Cd-induced changes in s ulfate content in roots (A

and C) and leaves (B and D) of B. napus. Plants were grown

hydroponically for 2 weeks and then transferred to the same

solution containing 0, 20, 40, 80, 120 £gM Cd for 72 h (A and B),

or 0

(

„T) and 40

(

„S) £gM Cd for 0, 6, 12, 24, 48, 72 and 96 h (C

and D). Vertical bars represent standard deviation of the mean

(n=3). Asterisks indicate that mean values are significantly dif-

ferent between the treatment and control (p<0.05).

Figure 9. Change in sulfide (A) and glutathione (B)

content in

roots and leaves of B. napus under Cd stress. Plants were grown

hydroponically for 2 weeks and then transferred to the same

solution containing 40 and 80 £gM Cd for 72 h. Vertical bars rep-

resent standard deviation of the mean (n=3). Asterisks indicate

that mean values are significantly different between the treat-

ment and control (p<0.05).

Figure 10. Changes in non-protein thiols (NP T) contents in

roots (A and C) and leaves (B and D) of B. napus. Plants were

grown hydroponically for 2 weeks and then transferred to the

same solution containing 0, 20, 40, 80, 120 £gM Cd for 72 h (A

and B), or 0 („T) and 40 („S) £gM Cd for 0, 6, 12, 24, 48, 72 and

96 h (C and D). Vertical bars represent standard deviation of the

mean (n=3). Asterisks indicate that mean values are significantly

different between the treatment and control (p<0.05).

SUN et al. ¡X Sulfate transporters in response to Cd

51

expression of BnSultr2;2 in different tissues indicates that

it is localized in the root and leaf of B. napus. This result

is consistent with the previous observations in A. thaliana,

where a cloned low-affinity sulfate transporter was also

expressed in roots and leaves (Takahashi et al., 2000).

Although we did not make a further identification of the

low-affinity sulfate transporter, the low phylogenetic

distance between BnSultr2;2 and AtSultr2;2 suggested that

they share a similar funtion.

Regulation of the sulfate transporter activity that is

dependent on S-nutritional status has been investigated

in a variety of plants (Hawkesford, 2003). Expression

of low-affinity sulfate transporter genes in response to

sulfur deficiency has been proposed to be behind im-

provements in S-utilization efficiency within plants

(Hawkesford, 2000). However, little is known about

the detailed expression pattern of LAST in response to

Cd exposure. It has been demonstrated that a gene for a

putative low-affinity sulfate transporter from B. juncea

exhibited a significantly reduced expression in roots upon

Cd

treatment (Heiss et al., 1999). We have likewise found

that the root BnSultr2;2 expression was progressively

down-regulated by Cd exposure (Figure 6A). In A.

thaliana, expression of AtSultr2;2 was detected in root

phloem and leaf vascular bundle sheath cells, suggesting

that the low affinity sulfate transporter AtSultr2;2 was

involved in the release of sulfate from xylem vessels to

palisade and mesophyll cells (Takahashi et al., 2000). In

the present study, the reduced expression of BnSultr2;2 in

Cd-treated roots indicates that the B. napus plants might

prevent a sulfate transfer from roots to shoots because

synthesis of PCs and other low or high-molecular-weight

complexes in roots requires sufficient sulfate (Heiss et al.,

1999), thus causing more sulfate to be retained in roots for

local assimilation. The result could be supported by the

increased sulfate content in root (Figure 8). On the other

hand, expression of BnSultr2;2 was up-regulated in leaf in

response to Cd exposure (Figure 6B), which is consistent

with the expression pattern under sulfur starvation (Figure

5B). The pattern of BnSultr2;2 expression above-ground

suggests Cd-induced short-term sulfur starvation, and

more sulfate uptake into leaf mesophyll cells may be re-

quired for sulfur-containing metabolite synthesis for metal

chelating.

To get an insight into the expressional pattern of

sulfate transporters in response to Cd stress, we simulta-

neously isolated and analyzed another gene encoding a

high affinity sulfate transporter (BnSultr1;1) in B. napus.

BnSultr1;1 was expressed only in roots, suggesting that

BnSultr1;1 belongs to the Group-1 high-affinity sulfate

transporters involved in the uptake of sulfate into roots

at low sulfate availability. BnSultr1;1 in roots showed a

base level expression (Figure 5C). However, both sulfur

starvation and Cd exposure resulted in a strong expression

o f BnSultr1;1 in roots (Figure 5C and 6C). Sulfate

starvation could lead to a decrease of GSH (Leustek

and Saito, 1999), and PC synthesis induced by Cd could

also lead to a transient depletion of GSH pools in roots

(Scheller, 1987). This depletion may be what increases the

uptake of sulfate. It was interesting to find a 12-h lag phase

between the Cd treatment and the increased BnSultr1;1

expression (Figure 7C). This suggested that before the on-

set of a considerable expression of BnSultr1;1 could occur,

time for cadmium to induce GSH depletion was required.

It has been proposed that both low- and high-affinity

sulfate transporters play key roles in sulfate uptake,

translocation, and distribution in higher plants (Buchner

et al., 2004b; Sun et al., 2005). However, the mechanism,

by which Cd influences the sulfur status and modifies

the gene expression of both group-1 and group-2 sulfate

transporters is not fully understood. To relate Cd-induced

alteration in the expression of BnSultr1;1 and BnSultr2;2

to sulfate uptake, accumulation, and assimilation in B.

napus, the total sulfate accumulation and other reduced

metabolite content was measured. Our results shows that

the accumulation of sulfate and sulfide in Cd-treated roots

increased significantly. Also, the level of glutathione

and non-protein thiols in the roots with Cd was found to

increase. The observed modifications of sulfate content

and increased metabolite production may reflect the local

effects of Cd in the organs or the Cd-driven demand

for sulfur nutrients for the whole plant. Indeed, the the

regulatory signals orginated from either the Cd-stressed

shoot or the treated roots. We noted that the B. napus

plants accumulated Cd rapidly upon Cd exposure (Figure

4). The accumulation long preceded the the enhanced

expression of BnSultr2;2, the sulfate accumulation rate,

and the synthesis of reduced products of sulfur. Taken

together, all of these results suggest an inter-organ

signal during Cd exposure responsible for sulfate uptake

regulation and reduction within different organs.

Acknowledgements. This study was supported by the

National Natural Science Foundation of China (No.

30070447)

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