sulfate reduction is increased in transgenic arabidopsis thaliana expressing 5′-adenylylsulfate...

Sulfate reduction is increased in transgenic Arabidopsisthaliana expressing 50-adenylylsulfate reductase fromPseudomonas aeruginosa

George Tsakraklides1, Melinda Martin1, Radhika Chalam1,y, Mitchell C. Tarczynski2, Ahlert Schmidt3 and

Thomas Leustek1,�

1Plant Biology and Pathology Department, Biotechnology Center for Agriculture and the Environment, Rutgers

University, New Brunswick, NJ 08901-8520, USA,2Pioneer Hi-Bred International Inc., A DuPont Company, Trait & Technology Development, 7300 NW 62nd Avenue, PO

Box 1004, Johnston, IA 50131-1004, USA, and3Institute for Botany, University of Hannover, Hannover, Germany

Received 2 August 2002; accepted 4 September 2002.�For correspondence (Tel. þ1 732 932 8165/ext. 326; fax þ1 732 932 0312; e-mail: [email protected]).yPresent address: Department of Molecular Biology, IDEC Pharmaceuticals, La Jolla, CA, USA.

Summary

The two-electron reduction of sulfate to sulfite in plants is mediated by 50-adenylylsulfate (APS) reductase,

an enzyme theorized to be a control point for cysteine synthesis. The hypothesis was tested by expression

in Arabidopsis thaliana under transcriptional control of the CaMV 35S promoter of the APS reductase from

Pseudomonas aeruginosa (PaAPR) fused with the rbcS transit peptide for localization of the protein to

plastids. PaAPR was chosen for the experiment because it is a highly stable enzyme compared with the

endogenous APS reductase of A. thaliana, and because PaAPR is catalytically active in combination with the

plant thioredoxins m and f indicating that it would likely be catalytically active in plastids. The results

indicate that sulfate reduction and O-acetylserine (OAS) production together limit cysteine synthesis.

Transgenic A. thaliana lines expressing PaAPR accumulated sulfite, thiosulfate, cysteine, c-glutamylcys-

teine, and glutathione. Sulfite and thiosulfate increased more than did cysteine, c-glutamylcysteine and

glutathione. Thiosulfate accumulation was most pronounced in flowers. Feeding of OAS to the PaAPR-

expressing plants caused cysteine and glutathione to increase more rapidly than in comparably treated wild

type. Both wild-type and transgenic plants accumulated sulfite and thiosulfate in response to OAS feeding.

The PaAPR-expressing plants were slightly chlorotic and stunted compared with wild type. An attempt to

uncover the source of thiosulfate, which is not thought to be an intermediate of sulfate reduction, revealed

that purified b-mercaptopyruvate sulfurtransferase is able to form thiosulfate from sulfite and b-mercapto-

pyruvate, suggesting that this class of enzymes could form thiosulfate in vivo in the presence of excess

sulfite.

Keywords: sulfate reduction, sulfite, thiosulfate, cysteine, c-glutamylcysteine, glutatnione, 50-adenylylsulfate,

O-acetylserine.

Introduction

Cysteine is an amino acid containing reduced sulfur in the

form of a thiol group. Its synthesis in plants can be divided

into three functional steps: uptake of sulfate into plant cells,

reduction of sulfate to sulfide, and assimilation of sulfide

into cysteine. Sulfate reduction is mediated by three

enzymes (Figure 1; Leustek and Saito, 1999). First, sulfate

is adenylated by ATP sulfurylase [EC 2.7.7.4] forming 50-

adenylylsulfate (APS). Sulfite is formed by APS reductase

[EC 1.8.4.9]. Then, sulfide is formed by sulfite reductase [EC

1.8.7.1]. The combined action of these three enzymes takes

sulfur from theþ6 to the �2 oxidation state. Assimilation

into cysteine occurs when sulfide reacts with O-acetylserine

(OAS) catalyzed by OAS thiol-lyase [EC 4.2.99.8](Figure 1).

OAS is formed by serine acetyltransferase [EC 2.3.1.30].

Chloroplasts contain all the enzymes needed for

cysteine synthesis. Isoforms of ATP sulfurylase, serine

The Plant Journal (2002) 32, 879–889

� 2002 Blackwell Publishing Ltd 879

acetyltransferase and OAS thiol-lyase are also localized in

the cytosol and serine acetyltransferase, and OAS thiol-

lyase isoforms are also localized in mitochondria.

Cysteine serves as the substrate for all the molecules in

plants containing �2 valance sulfur. These include a wide

range of compounds such as methionine, vitamins, co-

enzymes, secondary metabolites and proteins. The most

abundant of these are proteins and in cruciferous plants a

class of compounds termed glucosinolates. Normally, free

cysteine is a minor component of the total reduced sulfur

pool.

In order for the production of cysteine to be synchronized

with its utilization for growth, the sulfate reduction and

assimilation steps must be coordinated. This is, especially

important to avoid inappropriate accumulation of toxic

pathway intermediates including sulfite and sulfide. Both

of these intermediates are normally maintained at low

levels in plant cells due to their toxicity (Rennenberg,

1984). Several different mechanisms have been identified

for control of cysteine synthesis, including the regulation of

gene expression and regulation of enzyme activity. A wide

range of studies suggests that the key regulated steps are

sulfite formation by APS reductase, and OAS formation by

serine acetyltransferase (reviewed in Brunold and Rennen-

berg, 1997; Leustek et al., 2000). Using a transgenic plant

approach this model can be experimentally tested by ecto-

pic expression of specific enzymes. In the present study, the

effect of elevated APS reductase activity was examined by

expression in transgenic Arabidopsis thaliana of the APS

reductase from Pseudomonas aeruginosa.

There are two mechanisms for assimilative sulfate reduc-

tion in living organisms. APS reductases exist in plants and

in a wide range of bacteria including P. aeruginosa (Bick

et al., 2000; Setya et al., 1996) and others (Abola et al., 1999;

Neumann et al., 2000; Williams et al., 2002). A. thaliana

contains three genes encoding APS reductase, At1g62180,

At4g21990, and At4g04610 (Leustek, 2002). Another sulfate

assimilation pathway uses the enzyme 30-phospho-50-

adenylylsulfate (PAPS) reductase [EC 1.8.4.8], typified by

the founding member of this group, the CysH enzyme of

Escherichia coli. The prokaryotic assimilative APS reduc-

tases are proteins of approximately 29 kDa that utilize thior-

edoxin or glutaredoxin as an electron donor (Bick et al.,

2000). The plant APS reductases are approximately 49 kDa

and carry an additional polypeptide region at the carboxyl

terminus that is homologous with thioredoxin and func-

tions as an exclusive redox co-factor, mediating the cata-

lytic transfer of electrons from reduced glutathione (Bick

et al., 1998).

P. aeruginosa APS reductase was chosen for ectopic

expression in A. thaliana primarily because of its high

stability in vitro. By contrast, plant APS reductases are very

unstable enzymes (Suter et al. 2000). In addition, there is no

evidence for post-translational regulation of P. aeruginosa

APS reductase (unpublished observation) as there is for the

plant APS reductase (Bick et al., 2001). Lastly, P. aeruginosa

APS reductase was found to function well with plant-type

thioredoxins m and f (Bick et al., 2000) suggesting that it has

the potential to function in A. thaliana chloroplasts.

In the current study, expression of P. aeruginosa APS

reductase in A. thaliana (Figure 1) resulted in accumulation

of intermediates and products, consistent with the pre-

dicted effect of increased activity of the sulfate reduction

pathway and the hypothesis that APS reductase catalyzes a

regulated step in sulfate reduction.

Results

Preparation and characterization of transgenic A. thaliana

lines expressing P. aeruginosa APS reductase

A. thaliana was transformed by floral dipping with a DNA

construct intended for stable genome integration, consti-

tutive expression, and chloroplast targeting of PaAPR. The

seeds harvested after transformation were germinated on

kanamycin-supplemented medium for selection of trans-

formants. Surviving plants were transferred to potting mix

and grown for 45 days. Rosette leaves were tested for

PaAPR production by immunoblotting. The blots revealed

a number of lines that express an immunoreactive protein

of approximately 29 kDa, which is undetectable in the wild

type. Figure 2(a) shows a representative blot in which of 9 of

the 30 primary transformants were analyzed. Several

higher molecular weight proteins also cross-react weakly

with the antibody, however, they are observed in both the

wild-type and transgenic lines. The 29-kDa mass of the

specific immunoreacting protein is consistent with the

expected mass for PaAPR following proteolytic removal

of the transit peptide.

Figure 1. The pathway of reductive sulfate assimilation in A. thaliana. Thefigure shows that plant APS reductase may use reduced glutathione as anelectron source. Expression in transgenic plants of P. aeruginosa APSreductase (PaAPR), a thioredoxin-dependent enzyme (circled), could poten-tially increase sulfate assimilation.

880 George Tsakraklides et al.

� Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 879–889

Southern blot analysis was performed to identify plants

with single TDNA insertions from which homozygous lines

could be established for detailed biochemical analysis.

Plants 2.16, 2.5 and 3.2 were chosen. Heritable overexpres-

sion of PaAPR was detected in the progeny. Figure 2(b)

shows PaAPR expression in three individual plants from

homozygous lines established from each plant. Two of

them, lines 2.16 and 3.2 show high-level PaAPR expression,

whereas line 2.5 shows an intermediate level of PaAPR

expression.

By tailoring the conditions for measurement of APS

reductase it is possible to discern PaAPR activity from

endogenous APS reductase activity. A. thaliana APS reduc-

tase has a nearly absolute requirement for Na2SO4 in the

assay mixture (Table 1, line 1 versus line 2), whereas PaAPR

does not require Na2SO4. Under assay conditions opti-

mized for PaAPR (no Na2SO4 and using thioredoxin as an

electron donor) wild-type A. thaliana did not show activity,

whereas the three transgenic lines did show activity

(Table 1). Dividing the PaAPR activity by the endogenous

APS reductase activity observed in wild type revealed that

lines 2.16 and 3.2 have 7.1- and 7.8-fold greater total APS

reductase activity compared with wild type, than does lines

2.5, which has 2.9-fold more activity. It is not possible to

determine more accurately the total APS reductase activity

in transgenic plants because PaAPR has residual activity

under the conditions used to measure A. thaliana APS

reductase.

Comparing the relative levels of PaAPR activity with

PaAPR protein measured by immunoblotting showed a

close, but not identical match. Such a result could arise if

some of the PaAPR protein produced in A. thaliana is

inactive. It is important to note that in vitro activity is

measured under ideal conditions of unlimited substrates.

Therefore, it represents the potential maximum activity and

may not reflect the actual in vivo activity.

RNA blotting was used to further confirm PaAPR expres-

sion. Using a DNA probe that hybridizes with both the rbcS

and rbcStp–PaAPR transcripts, the blot in Figure 3 shows

that the transgenic lines produce an approximately 1200-

nucleotide transcript corresponding to rbcStp–PaAPR that

is not produced in wild type. An approximately 800-nucleo-

tide rbcS transcript is detected in both transgenic and wild-

type lines. Lines 2.16 and 3.2 express relatively more

rbcStp–PaAPR transcript than does line 2.5, revealing that

mRNA level correlates with the level of PaAPR protein

measured by immunoblotting and enzyme activity. More-

over, the abundance of rbcS mRNA is the same as in wild

type, therefore, the rbcS transcript can serve as an RNA-

loading control in the experiment.

Table 1 Correlation between APS reductase activity and PaAPRlevel in transgenic plants

SampleAPR or PaAPR activitynmol min�1mg protein�1

PaAPR proteinrelative values

wt 0.77a� 0.09 0wt 0b –2.16 5.44b� 0.03 3.0� 0.0.42.5 2.25b� 0.04 1� 0.53.2 6.02b� 0.38 4.3� 0.4

APS reductase assays were performed on protein extracts from20-day-old shoots of wild-type and homozygous transgenic A.thaliana lines expressing PaAPR. The assays were performedusing a reaction mixture described in Experimental Procedurescontaining either, a0.5 M sodium sulfate and 10 mM DTT orb25 mM E. coli thioredoxin and 10 mM DTT. Under the laterconditions endogenous plant APS reductase (APR) is inactive,whereas PaAPR is active. Three separate plants from each linewere measured and the mean is presented� SD. Although PaAPRand APR have differing reaction requirements, APR activitycannot be clearly distinguished alone in a plant extract containingsignificantly greater amounts of PaAPR activity, because PaAPRhas residual activity under the (a) reaction conditions. RelativePaAPR protein level was calculated, measuring the signalintensity of bands on the immunoblot of Figure 4 with aFluorChemTM imaging system (Alpha Innotech Inc., Mississauga,Ontario).

Figure 2. Immunoblot of PaAPR protein expression in transgenic A. thali-ana.(a) Initial screening of primary transgenic individuals. Lane 1 shows theresult with a wild-type plant, whereas lanes 2–10 show the expression ofPaAPR in individual transgenic plants designated 3.3, 3.1, 2.6, 2.5, 2.2, 1.9,1.7, 1.4, and 1.3. Samples were from leaves of 45-day-old plants.(b) Immunoblot of 20-day-old T3 homozygous individuals. Three separateplants from each line were analyzed.

Figure 3. PaAPR transcript in transgenic plants. The leaves of three indivi-dual 20-day-old homozygous plants were analyzed from each transgenicline. Sample identifications are at the bottom of each lane. The larger mRNA-labeled PaAPR is the transcript of the rbcS transit peptide–PaAPR fusionconstruct (rbcStp–PaAPR) and the smaller mRNA is rbcS. Both mRNAs canbe detected on the same blot because the respective probes do not interferewith hybridization of the other.


Deregulation of Sulfate assimilation in Arabidopsis thaliana 881

PaAPR plants of all three lines demonstrated a delayed

germination phenotype and they produced smaller leaves

that were slightly yellow compared with wild-type (not

shown). Growth stunting is evident as reduced fresh and

dry weight, but the ratio of fresh to dry weight was not

altered (Table 2). The transgenic lines flowered at the same

time as wild type, but fertility was slightly reduced.

Although flowers appeared normal, in some cases siliques

aborted.

Expression of P. aeruginosa APS reductase in A. thaliana

increases sulfate reduction and assimilation

The effect of PaAPR expression on the level of sulfur

metabolites was examined by acid extraction of plant

tissues and liquid chromatographic resolution and quanti-

fication of adducts resulting from reaction with mono-

bromobimane. The focus was on the thiols, cysteine,

g-glutamylcysteine, and glutathione and on inorganic

reduced forms of sulfur. The rosette leaves of vegetative,

20-day-old transgenic plants showed cysteine, glutathione,

and g-glutamylcysteine ranging from 150% to 200% of the

wild-type level (Figure 4). The increase in glutathione was

independently confirmed using the GSSG reductase-recy-

cling assay. A far greater increase was observed in a peak

co-eluting with sulfite, which was approximately 320% of the

wild-type level,andasecondpeakco-elutingwith thiosulfate,

which was approximately 360% of the wild-type level.

Organic and inorganic reduced sulfur compounds also

accumulated in various tissues of flowering stage, 32-day-

old plants (Figure 5). The levels of glutathione, g-glutamyl-

cysteine, and cysteine were higher in the transgenic plant

tissues than in wild type. The levels of glutathione and

cysteine were much higher in flowers and siliques than

in vegetative tissues for both wild-type and transgenic

plants. Interestingly, the level of g-glutamylcysteine was

very high in siliques compared with all other tissues ana-

lyzed. The increase of thiosulfate in the flowers of trans-

genic plants compared with the level in wild type ranged

from 211% of wild type in line 2.5 and 1100% in line 2.16.

Immunoblot analysis showed that PaAPR level was

very similar in all tissues of the transgenic plants when

normalized to total protein. This finding suggests that the

tissue-specific differences in levels of reduced sulfur com-

pounds may result from factors other than PaAPR activity

in the tissues.

Confirmation of the sulfite and thiosulfate measurements

and possible origin of thiosulfate in PaAPR-expressing

plants

Sulfite and thiosulfate have not previously been reported to

be present in normally grown plants. To confirm the iden-

tity of these compounds in PaAPR-expressing A. thaliana

their co-elution with standard sodium sulfite and sodium

thiosulfate was established using three chromatographic

methods differing in pH and composition of the mobile

phase (see Experimental procedures). Typical chromato-

grams of wild type and a PaAPR-expressing transgenic

plant are presented in Figure 6 showing the peaks asso-

ciated with the monobromobimane adducts of the major

Table 2 Effect of PaAPR expression on fresh and dry weight of20-day-old plants

WT Line 2.16 Line 3.2 Line 2.5

Fresh weight (mg) 149� 15 106� 17 81� 8 97� 17Dry weight (percentageof fresh weight)

7.9� 0.4 7.0� 0.5 7.7� 0.4 7.6� 0.5

Entire 20-day-old rosettes were harvested and weighed. Dryweight is represents the average of three plants. Dry weight wasmeasured after drying at 558C for 18 h.

Figure 4. Reduced sulfur compounds in the shoot of 20-day-old plants. Allvalues are given in pmol mg�1 FW. The mean value from three individualplants is presented�SD. Values denoted with different letters are signifi-cantly different at P¼ 0.05.



organic thiol compounds, sulfite and thiosulfate. The chro-

matograms show that the sulfite and thiosulfate adducts

elute from the reverse phase column as sharp, symmetrical

peaks.

The identity of sulfite and thiosulfate was further con-

firmed by adding plant extracts de-proteinized with HCl to

rhodanese or sulfotransferase enzymatic assays and obser-

ving the appearance or disappearance of thiosulfate and

sulfite from the chromatogram. Thiosulfate serves as a

substrate for b-mercaptopyruvate sulfurtransferase, for-

merly known as rhodanese, which forms thiocyanate from

cyanide and thiosulfate according to the following equa-

tion.

S� SO�3 þ CN ! SCNþ SO2�3

Reaction of transgenic plant extracts with cyanide and

purified, re-combinant A. thaliana b-mercaptopyruvate

sulfurtransferase derived from the MST1 gene (locus

At1g79230, Papenbrock and Schmidt, 2000) resulted in the

MST1-dependent disappearance of the chromatographic

peak linked with thiosulfate (not shown). Bovine liver

rhodanese was equally effective at removing thiosulfate.

The ability to form thiosulfate from sulfite by b-mercap-

topyruvate sulfurtransferase has not previously been

reported, although such an activity was hypothesized

(Papenbrock and Schmidt, 2000). Table 3 shows that in a

Table 3 Formation of thiosulfate by purified thiosulfatesulfurtransferase from A. thaliana

Sulfite added (nmol) Thiosulfate formed (nmol)

0 010 14.425 16.750 32.9

100 85.7250 112.4500 123.9

1000 124.0

The assay conditions were as following: each assay contained in atotal volume of 0.5 ml the following compounds: 10 mmol Hepes-KOH, pH 8.3, 0.5 mmol b-mercaptopyruvic acid, 2.5 mmol b-mer-captoethanol, sulfite as indicated, and 0.9 mg b-mercaptopyruvatesulfurtransferase purified as in Papenbrock and Schmidt (2000).Incubation was for 30 min at 378C. Reactions were terminated,reacted with monobromobimane and analyzed by HPLC asdescribed in the Experimental procedures.

Figure 5. Reduced sulfur compounds in tissues from 32-day-old floweringstage plants. Rosette leaf, stem, cauline leaf, flower and silique tissue wasseparately analyzed. The mean value from three individual plants is pre-sented�SD. Values denoted with different letters are significantly differentat P¼0.05. samples within each group were statistically compared.

Figure 6. HPLC chromatograms comparing reduced sulfur compounds inwild-type and PaAPR-expressing A. thaliana. The chromatograms areshown at the same scale, the one from the PaAPR-expressing plant lineis offset to show the differences from wild type. The identity of peakscorresponding to specific reduced sulfur compounds is indicated on theplots. The chromatograms were derived from analysis of flower tissue ofwild-type and transgenic line 2.16.



defined in vitro system thiosulfate formation can be mea-

sured with as little as 10 mM sulfite, the lowest level that was

tested. The apparent Km for sulfite is 0.36 mM. These results

indicate that A. thaliana b-mercaptopyruvate sulfurtransfer-

ase has the potential to form thiosulfate in vivo. It should

also be noted that there are at least 11 genes in A. thaliana

related to sulfurtransferases. Some of these could produce

a protein that is much more active with sulfite as the

substrate.

An important note about the sulfite measurements

reported here is that it is an underestimate of the actual

level in the PaAPR-expressing A. thaliana tissues. For tech-

nical reasons, it was necessary to extract plant samples

under acidic conditions and with the inclusion of DTT as a

reductant of cysteine and glutathione. However, both sul-

fite and thiosulfate are acid labile and sulfite reacts with

DTT, which leads to losses. For thiosulfate, the losses were

mitigated by rapid sample extraction and alkylation with

monobromobimane. Rapid sample processing was insuffi-

cient to curtail the loss of sulfite, which was recovered at

approximately 35%, or sulfide, which was not measured.

Thus, the values for reduced inorganic sulfur compounds

reported here should be interpreted with the understanding

that the analytical method was a compromise necessary for

the measurement of all the metabolites.

The effect of OAS feeding on the level of organic

and inorganic reduced sulfur compounds in

PaAPR-expressing plants

The accumulation of reduced sulfur intermediates in trans-

genic A. thaliana expressing PaAPR suggests that the rate

of sulfur reduction has been increased. The accumulation of

inorganic sulfur compounds is greater than is the accumu-

lation of cysteine, suggesting that OAS synthesis may limit

the incorporation of reduced inorganic sulfur into cysteine

in the transgenic plants. To assess this question the effect of

feeding OAS in the growth medium on cysteine synthesis

was determined. Wild-type and transgenic plants grown for

13 days on agar medium were transferred to medium con-

taining 0.5 mM OAS and the levels of cysteine, glutathione,

sulfite and thiosulfate were measured. Both wild-type and

PaAPR-expressing plants accumulated cysteine after OAS

feeding (Figure 7a). The accumulation of cysteine in the

transgenic lines 2.16, 3.2, and 2.5 peaked at 20 h and

remained constant thereafter (only the data for 2.16 is

shown in Figure 7a), whereas the wild type showed a steady

increase up to 74 h. The initial rate of cysteine accumulation

was higher for all the transgenic lines compared with wild

type (Figure 7b). The kinetics of glutathione increase fol-

lowed a different pattern. Both the wild-type and transgenic

lines showed similar rates of glutathione accumulation

after OAS feeding (Figure 7c). Interestingly, thiosulfate

(Figure 7d) and sulfite (not shown) also increased in abun-

dance after OAS feeding in both the transgenic lines and

wild-type. Several points should be noted with regard to the

OAS feeding experiment. All the PaAPR-expressing plants

behaved very similarly to line 2.16 for which data is shown

Figures 7(a,c,d). Secondly, under the in vitro growth condi-

tions used for this experiment, the levels of cysteine and

glutathione are lower for PaAPR-expressing and wild-type

plantscomparedwithplantsgrowninpottingmix.This could

be due to the younger age of the plants and to the differing

growth conditions in vitro compared with in potting mix.

Figure 7. Effect of O-acetylserine (OAS) feed-ing. The results from experiments with wildtype and line 2.16 are presented.(a) Time course of cysteine accumulation.(b) Comparison of cysteine concentration inuntreated and 20 h OAS-treated samples. Thefold change in cysteine was calculated relativeto the level in the sample not exposed to OAS.(c) Time course of glutathione accumulation.(d) Time course of thiosulfate accumulation.The mean value from three individual plants ispresented�SD. Values denoted with differentletters are significantly different at P¼0.05. Linesymbols in graphs (a), (c), and (d) represent: (*)wild type not exposed to OAS (*) wild typeexposed to 0.5 mM OAS (!) line 2.16 exposedto 0.5 mM OAS. The representations of data ingraph B are indicated directly on the bars.



Discussion

The primary conclusion of the experiments reported here is

that expression of P. aeruginosa APS reductase in A. thali-

ana results in an increase in sulfate reduction leading to the

accumulation of reduced inorganic and organic forms of

sulfur. The result confirms that sulfite formation is a limit-

ing step in the assimilative sulfate reduction pathway of

A. thaliana.

Normally, cysteine synthesis is regulated by coordinated

production of sulfide and OAS achieved by a range of

mechanisms including the regulation of gene expression

and post-translational control of enzyme activity. By

expressing P. aeruginosa APS reductase in A. thaliana

the two processes have been uncoupled, due in part to

constitutive expression driven by the 35S promoter and

perhaps to the catalytic properties of PaAPR. Uncoupling of

sulfide production from OAS production resulted in accu-

mulation of cysteine, g-glutamylcysteine, and glutathione

as well as the accumulation of sulfite and thiosulfate, a

metabolite that likely is formed in direct response to sulfite

accumulation.

It was previously reported that expression of E. coli

serine acetyltransferase (SAT) in either tobacco or potato

resulted in accumulation of cysteine and derived metabo-

lites (Blaszczyk et al., 1999; Harms et al., 2000). This

phenomenon was explained by the dual role that OAS

is known to play as the substrate for cysteine synthesis

and as a regulator of expression of sulfate assimilation

genes. Application of OAS to plants induces expression of

sulfate transporter and APS reductase resulting in accu-

mulation of cysteine (Koprivova et al., 2000; Smith et al.,

1997), just as was confirmed here (see Figure 7). Presum-

ably, sulfate reduction was also up-regulated in the plants

expressing E. coli SAT due to endogenous OAS overpro-

duction. The enhanced production of cysteine resulting

from PaAPR expression suggests that sulfite reduction is

also a rate limiting reaction. Stimulation of cysteine pro-

duction by exposing plants to H2S, which would theore-

tically have a similar effect as APS reductase up-

regulation, has been reported in Pisum sativum (von

Arb and Brunold, 1986) and OAS is known to limit the

in vivo rate of cysteine synthesis in Brassica oleracea

fumigated with H2S (Buwalda et al., 1993). One possible

explanation for this could be that OAS negatively regulates

SAT via a post-translational mechanism (Droux et al.,

1998). Endogenous synthesis of H2S or exposure to it

would stimulate consumption of OAS into cysteine, result-

ing in a decline in free OAS level, thereby stimulating SAT

activity by removal of the inhibiting agent. Nonetheless,

the fact that PaAPR-expressing plants accumulate sulfite/

thiosulfate and that the rate of cysteine synthesis is

greater than wild type after exogenous feeding of OAS,

suggests that endogenous OAS production does not keep

pace with the rate of sulfate reduction in the transgenic

plants.

Quantitative analysis of the PaAPR-expressing transgenic

plants revealed that the accumulation of reduced sulfur

compounds was not proportional to the level of PaAPR

activity. There are several possible explanations for this

result. One is that PaAPR is not fully functional under the

conditions in the stroma. A second possibility is that either

ATP sulfurylase or sulfite reductase activity limits the rate of

sulfate reduction in the transgenics. A third possibility is

that degradation or metabolism, participate in limiting the

level of reduced sulfur compounds. Finally, with regard to

sulfite and thiosulfate, the chemical reactiveness of these

compounds suggests that adducts could form in vivo which

would not be detectable with the assay procedure used

here.

The first possibility has interesting implications because

of the different substrate requirements for PaAPR com-

pared with endogenous A. thaliana APS reductase. PaAPR

relies on reduced thioredoxin as an electron donor (Bick

et al., 2001), whereas plant APS reductase uses glutathione

(Bick et al., 1998). Although PaAPR can efficiently use

plastid forms of thioredoxin in vitro (Bick et al., 2001),

the in vivo concentration of reduced thioredoxin may be

limiting. Plastid thioredoxins are reduced by ferredoxin-

dependent thioredoxin reductase only upon illumination of

chloroplasts and are used primarily for regulation of the

dark reactions of photosynthesis (Buchanan, 1991). Endo-

genous A. thaliana APS reductase may not be exposed to

the same limitation because it is thought to utilize reduced

glutathione as an electron donor (Bick et al., 1998), and the

ratio of oxidized to reduced glutathione is thought to be

diurnally constant.

The possibility that other steps in sulfate reduction limit

flux of the pathway must also be considered. ATP sulfur-

ylase overexpression increases assimilative sulfate reduc-

tion in Brassica juncea (Pilon-Smits et al., 1999), suggesting

that it is a limiting enzyme in this species. ATP sulfurylase

overexpression in tobacco had no effect, suggesting that it

is not limiting in this species (Hatzfeld et al., 1998). Whether

ATP sulfurylase is limiting in A. thaliana has not yet been

explored. Sulfite reductase could be another limiting

enzyme.

The possibility that metabolism or degradation pathways

influences the level of reduced sulfur compounds arises

because degradative enzymes have been identified in A.

thaliana. Two genes potentially encoding cysteine desulf-

hydrase have been identified, encoded by the loci

At1g08490 and At5g65720. Cysteine desulfurydrase, which

converts cysteine into pyruvate, ammonia, and elemental

sulfur, is implicated in production of iron–sulfur centers

(Zheng et al., 1993). Recently, A. thaliana sulfite oxidase, an

enzyme that converts sulfite to sulfate, has been cloned and

the enzyme characterized (Eilers et al., 2001). Very high



sulfite oxidase activity has been reported in certain plant

species (Jolivet et al., 1995). If sulfite oxidase activity is

similarly high in A. thaliana, a futile cycle could exist in

PaAPR-expressing plants, in which the rates of sulfate

reduction and sulfite oxidation exist in an equilibrium.

b-Mercaptopyruvate sulfurtransferase, formerly known as

rhodanese, and recently characterized from A. thaliana

(Papenbrock and Schmidt, 2000), is capable of a variety

of sulfur transfer reactions. One activity is the transfer of

the thiol group of thiosulfate to cyanide to form thio-

cyanate, an activity that was used here to confirm the

identity of sulfite and thiosulfate in the HPLC method used

for thiol analysis. Another activity, reported for the first

time in this paper, is the ability of A. thaliana b-mercapto-

pyruvate sulfurtransferase to produce thiosulfate in vitro

in the presence of sulfite (see Table 3). This finding

provides a possible route for the synthesis of thio-

sulfate in PaAPR-expressing plants by sulfurtransferases

(Papenbrock and Schmidt, 2000). The finding of thiosulfate

formation activity by b-mercaptopyruvate sulfurtransferase

may also explain the results of Sorbo (1957), who first

identified thiosulfate formation in a cell-free system. There

appear to be at least 11 genes in A. thaliana with homology

to b-mercaptopyruvate sulfurtransferases (At1g09280,

At1g16460, At1g79230, At2g17850, At2g40760, At2g42220,

At3g08920, At4g24750, At4g27700, At5g66040, At5g66170).

Inthefuture, itwillbeinformativetodeterminewhetheranyof

the enzymes derived from these genes are specialized for

thiosulfate metabolism.

The finding that thiosulfate is formed in PaAPR-expres-

sing plants is interesting because thiosulfate was postu-

lated 32 years ago to be an intermediate in the plant

assimilative sulfate reduction pathway. Thiosulfate forma-

tion from sulfate was observed in vitro from extracts of

Chlorella pyrenoidosa1 (Levinthal and Schiff, 1968).

Although thiosulfate could not be measured in living,

wild-type C. pyrenoidosa the mutant Sat2�, lacking a func-

tional sulfite reductase, was found to produce thiosulfate

from sulfate (Hodson et al., 1971). This finding may now be

understood in the context of the current report. Thiosulfate

is likely to have been produced in Sat2� C. pyrenoidosa by a

mechanism similar to that operating in PaAPR-expressing

A. thaliana plants. Namely, that both systems accumulate

sulfite, which may be converted to thiosulfate by a sulfur-

transferase. Whereas, PaAPR-plants overproduce sulfite

due to increased APS reductase activity, in Sat2� C. pyr-

enoidosa sulfite accumulates due to a lack of sulfite reduc-

tase.

Experimental procedures

Plant growth conditions

Plants were grown in lighted growth chambers in either of twodifferent ways. They were grown under axenic conditions in Petridishes on Murashigi and Skoog nutrients (Life Technologies,Gaithersberg, MD) provided at half the concentration recom-mended by the supplier and solidified with 8 g l�1 agar, or theywere grown in pots on peat-based PRO-MIX BX (Premier Horti-culture Inc., Dorval, Quebec) fertilized at each watering with Jack’sClassic Blossom Booster 10 : 30 : 20 (N:P:K, J.R. Peters Inc., Allen-town, PA) dissolved in ultrapure water at a concentration of 0.5g l�1 and adjusted to pH 6.2–6.5 with KOH. Growth chamber condi-tions were 16 h light at an intensity of 190 mmol photons m�2 sec�1

and 8 h darkness maintained at 248C in the light and 208C in thedark.

In some experiments, OAS was exogenously supplied to A.thaliana plants. A 100-mM stock solution of OAS dissolved inwater was adjusted to pH 4.4 with KOH and was filter sterilizedand added at 0.5 mM to autoclaved Murashigi and Skoog mediumafter cooling to 558C. In OAS treatment experiments, plants weregerminated and grown for 13 days on OAS-free medium in Petridishes oriented vertically to allow the roots to grow on the agarsurface. The plants were then transferred to OAS-containing med-ium for periods specified in the figure legends.

Construction of the transformation vector for expression

of P. aeruginosa APS reductase gene in A. thaliana

chloroplasts, plant transformation, and establishment

of homozygous lines

The coding sequence of the P. aeruginosa APS reductase wasfused in-frame with the coding sequence of the A. thaliana rbcStransit peptide (rbcStp) using PCR in a manner analogous to thatdescribed in Tomme et al. (1995). rbcStp was amplified frompGS1400 (De Almeida et al., 1989) with primers A (50-GGGGGATCCTGACCAAAGCACTAGA-30) and B (50-AATGGTAG-CAAAGGGCATGCAGTTAACTCTTCC-30). The bold letters in primerA correspond to the region of the rbcS gene from �36 to �51upstream of the translation initiation site and the remaining letterscorrespond to an added BamHI site. The APS reductase codingsequence was amplified from pUC–PaAPR (Bick et al., 2000) withprimers C (50-GGAAGAGTTAACTGCATGCCGCCCTTTGCTACCA-TT-30) and D (50-GGGGAGCTCAGGCCTTGCTGATCAGGTT-30). Pri-mers B and C are complementary to each other. The bold letters inthese primers correspond to the first six codons of APS reductaseand the remaining letters to the last five codons of rbcStp. The boldletters in primer D correspond to the last six codons and transla-tional stop codon of APS reductase and the remaining letters to anadded SstI site. The fusion was cloned with BamHI and SstI intoEL103, a pBI121 derivative carrying the tobacco mosaic virusomega translational enhancer sequence (Mittler and Lam, 1995).The fusion product was sequenced and confirmed to be identicalto rbcStp reported by De Almeida et al. (1989) and APS reductasecoding sequence reported in Bick et al. (2000).

A. thaliana Col-7 (Arabidopsis Biological Resource Center, stocknumber CS3731) was transformed by the floral dip method usingAgrobacterium tumefaciens strain GV3101 carrying the plasmidMP90. Four individual plants were transformed and the seedproduced by each were collected into separate pools. Primarytransformants selected on agar medium containing 50 mg/ml kana-mycin were transferred to soil, analyzed for PaAPR expression and

1It should be noted that the reductant used by Levinthal and Schiff (1968),2,3-dimercaptopropan-1-ol, spontaneously forms thiosulfate in a chemicalreaction with sulfite. Thus, the original description of thiosulfate formationin a plant extract was not the result of an enzyme activity.



allowed to self-pollinate. Kanamycin-resistant, non-segregatinglines were established by screening the progeny of self-pollinatedplants. Detailed physiological characterization was performed onhomozygous lines, defined as non-segregating, kanamycin-resis-tant lines carrying a single-transgene construct.

Immunoblotting

Samples harvested for immunoblotting included leaves of 20-day-old plants or leaves, inflorescence stems, flowers, siliques, andcauline leaves from 32-day-old plants. The tissues were ground in100 mM Tris–HCl pH 8.0, the lysate was centrifuged at 14 000 g at48C, and the protein concentration was determined in the extractusing the Bio-Rad Protein AssayTM reagent (Bio-Rad Inc., Her-cules, CA). For immunoblotting, 10 mg protein was electrophoresedby SDS-PAGE, blotted onto Immobilon-PTM membrane (MilliporeInc., Bedford, MA), and the membrane processed using polyclonaa ntiserum raised against PaAPR used at a dilution of dilution 1:2000. Immune complexes were detected by chemiluminescenceusing the RenaissanceTM Kit (Perkin-Elmer Life Sciences, Boston,MA).

For antibody production, PaAPR protein was produced from aHis-tag fusion construct of GenBank accession U95379 (Delic-Attreet al., 1997) from nucleotide 530 to nucleotide 1330. Polyclonalantibodies were produced as described by Woo et al. (2001).

RNA blotting

RNA was extracted from whole, 20-day-old plants, using TRI-ZOLTM (Life Technologies Inc., Gaithersberg, MD). RNA sampleswere electrophoresed on a 1% (w/v) agarose, denaturing formal-dehyde gel, and transferred to ZetaProbe membrane (Bio-Rad Inc.)using a pressure blotter. The membrane was processed asdescribed by the manufacturer. A DNA probe consisting of therbcStp–PaAPR fusion was used to detect RNA transcripts for bothrbcS and rbcStp–PaAPR. A DNA probe consisting of the PaAPRcoding sequence was used to detect only the rbcStp–PaAPR tran-script. DNA probes were labeled using the Random Primers DNALabeling System (Life Technologies) with [a-32P]dCTP (111 TBqmmol�1). Blots were exposed to film.

Enzyme assays

Plant extracts were prepared as described above in buffer contain-ing 100 mM Tris–HCl, pH 8.0, and 0.5 M sodium sulfate for mea-surement of A. thaliana APS reductase activity or 100 mM Tris–HCl,pH 8.0, for measurement of PaAPR activity. The assay mixture formeasurement of plant APS reductase included 50 mM Tris–HCl(pH 8.5), 1 mM EDTA, 500 mM Na2SO4, 25 mM [35S]APS (specificactivity 500 Bq nmol�1), 10 mM DTT and protein extract. Sodiumsulfate was omitted from the assay mixture for measurement ofPaAPR and 25 mM E. coli thioredoxin (Promega Corp., Madison, WI,cat. no. Z7051) was added. Both assays were incubated at 308C for20 min and processed as described by Bick et al. (2000). Thediffering extraction and assay conditions were designed to differ-entiate A. thaliana APS reductase from PaAPR. The plant enzymeshows very low activity when assayed in the absence of Na2SO4

(Schmidt, 1975), whereas PaAPR is fully active without the pre-sence of Na2SO4 and its activity is stimulated approximately four-fold by the addition of thioredoxin to the assay mixture (Bick et al.,2000). ATP sulfurylase was measured as described by Lee andLeustek (1999).

In vitro thiosulfate formation by b-mercaptopyruvate

sulfurtransferase

The thiosulfate formation ability of b-mercaptopyruvate sulfur-transferase was assayed in 0.5 ml containing 10 mmol HepesKOH(pH 8.3), 0.5 mmol b-mercaptopyruvic acid, 2.5 mmol b-mercap-toethanol, and either 10 mmol KCN or 2.5 mmol sulfite, and A. thali-ana MST1. MST1 was produced in E. coli and purified as describedby Papenbrock and Schmidt (2000). Enzymatic reactions wereterminated by the addition of acetonitrile. Products and substrateswere resolved by HPLC and quantitated as described below.

Measurement of reduced sulfur compounds and organic

thiols

Plant samples were harvested at the times specified in the figurelegend for OAS feeding experiments or 2 h after onset of the lightperiod in all other experiments. Fresh weight was recorded and thesamples immediately frozen and stored at �808C until analysis.Frozen tissue was extracted with 0.1 M HCl and 1 mM EDTA at10 ml mg�1 FW by grinding with a mortar and pestle. The extractswere centrifuged at 15 000 g at 58C and the supernatant recovered.Eighty microliters of extract was adjusted to pH 8.0 with 10 ml 0.5 M

sodium borate, pH 9.5. Five microliters of 30 mM DTT was addedand the mixture incubated for 10 min at room temperature toassure that all thiols were reduced. Then, 5 ml of 60 mM mono-bromobimane dissolved in acetonitrile was added and the mixtureincubated in the dark at room temperature for 20 min. Afterwards,100ml of 20% (v/v) acetic acid were added to reduce the pH to about3.0, a pH at which the monobromobimane adducts are more stable.

The monobromobimane adducts were resolved by liquid chro-matography using a Beckman System Gold 2690 (Beckman Coul-ter, Fullerton, CA) separation module equipped with a 126 solventmodule connected to an autosampler and an HITACHI F1080fluorescence detector (Danbury, CT) and controlled with Beckman32-Karat software or on a Waters Corp. (Milford, MA) Alliance LCSystem equipped with Millenium32 software, a Model 2690Separation Module, and a Model 474 fluorescence detector.Chromatography was performed at 358C on the Beckman systemand 328C on the Waters system with a Nova-Pak C18 SentryGuard column (3.9 mm 20 mm) followed in series with a3.9 mm 150 mm Waters AccQ-Tag column. The monobromobi-mane adducts were measured with the fluorescence detector set toan excitation wavelength of 360 nm and an emission wavelengthof 450 nm. Eluent A, containing sodium acetate and triethylamineat pH 5.05, was purchased as a concentrate from Waters. Thecomposition of eluent B was acetonitrile:water (30 : 70). The elutionmethod was 0–9 min, 6% B; 9–16 min, linear gradient to 8.5% B; 16–22 min, linear gradient to 25% B; 22–30 min, linear gradient to 100%B at a flow rate of 1.5 mL min�1. Standard curves were establishedusing the following chemicals: L-cysteine (Fluka catalog #30089),glutathione (Calbiochem catalog #3541), g-glutamylcysteine,sodium sulfite, sodium thiosulfate (from Sigma Chemical Co.,catalog numbers G-3903, S-5050, and S-1648, respectively).

Since sulfite and thiosulfite have not previously been reported toaccumulate in plant tissues, the co-migration of the A. thalianametabolites with these standards was confirmed using two addi-tional chromatographic methods. For method 2, the stationaryphase was the same as in method 1, but temperature was main-tained at 358C and flow rate was 1.0 ml min�1. Mobile phase A,contained 0.25% acetic acid, pH 4.3 with NaOH; mobile phase Bwas methanol:water (65 : 35). Samples were loaded to the columnequilibrated in 100% mobile A. The elution method was 0.5–1 min,



linear gradient to 8% B; 1–12 min, linear gradient to 11.5% B; 12–20 min, linear gradient to 18% B; 20–22 min linear gradient to 40%B; and 22–24 min linear gradient to 100% B. For method 3, sta-tionary phase was the same as in method 1, temperature wasmaintained at 258C and flow rate was 1.0 ml min�1. Mobile phase Acontained 0.25% acetic acid at pH 3.5 with NaOH; mobile phase Bwas methanol:water (65 : 35). Samples were loaded to the columnequilibrated in 13% B in 87% A. The elution method was 0–9 min13% B; 9–16 min a linear gradient to 50% B; 16–17 min, lineargradient to 100% B; and 17–25 min 100% B.

Recovery of reduced sulfur compounds and organic thiols wasdetermined as follows. Physiological amounts of cysteine, glu-tathione, g-glutamylcysteine, thiosulfate or sulfite were added toleaf samples from wild-type A. thaliana. The spiked samples wereimmediately extracted and analyzed as described above. Recoverywas estimated at greater than 95% for cysteine, glutathione, g-glutamylcysteine, thiosulfate, and approximately 35% for sulfite.

Confirmation of the identity of thiosulfate in plant extracts wascarried out using either Bovine liver rhodanese (Sigma Chemical,Corp., R 17756) or A. thaliana MST1. The reaction contained in0.05 ml, 1 mmol HepesKOH (pH 8.3), 1 mmol KCN, 0.25 mmol b-mer-captoethanol, three units bovine rhodanese or 9 mg A. thalianaMST1, and 1 mmol thiosulfate (as an activity control) or a neutra-lized acid extract of A. thaliana. The reaction was incubated for 2 hat 378C. Thiosulfate was measured by HPLC of the monobromo-bimane adduct as described above. The neutralized acid extract ofA. thaliana was also prepared exactly as described above.

Glutathione was also measured using a modification of the 5,50-dithiobis(2-nitrobenzoic acid (DTNB)) GSSG reductase recyclingassay adapted to a microtiter plate format (Anderson, 1985).

Statistical analysis

Data derived from three independent experiments were analyzedby two-component analysis of variance using the statistics func-tion of Excell 2000 (Microsoft Corporation).

Acknowledgements

This work was funded in part by grants from the United StatesNational Science Foundation grants IBN-9817594 (TL) and MCB-0094062 (MM), the German VW-Stiftung (AS), and Pioneer Hi-BredInternational Inc. The authors wish to express their appreciation toDr Michael Timko for providing pGS1400.

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