The immediate-early ethylene response gene OsARD1 encodes an acireductone dioxygenase involved in...

The immediate-early ethylene response gene OsARD1encodes an acireductone dioxygenase involved inrecycling of the ethylene precursor S-adenosylmethionine

Margret Sauter1,*, Rene Lorbiecke2, Bo OuYang3, Thomas C. Pochapsky3,4 and Guillaume Rzewuski1

1Botanisches Institut, Universitat Kiel, Olshausenstrasse 40, 24098 Kiel, Germany,2Biozentrum, Universitat Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany,3Departments of Chemistry and4Biochemistry, Brandeis University, 415 South Street, Waltham, MA 02454-9110, USA

Received 1 July 2005; revised 15 August 2005; accepted 23 August 2005.*For correspondence (fax þþ49 431 880 4222; e-mail [email protected]).

Summary

Methylthioadenosine (MTA) is formed as a by-product of ethylene biosynthesis from S-adenosyl-L-methionine

(AdoMet). The methionine cycle regenerates AdoMet from MTA. In two independent differential screens for

submergence-induced genes and for 1-aminocyclopropane-1-carboxylic acid (ACC)-induced genes from

deepwater rice (Oryza sativa L.) we identified an acireductone dioxygenase (ARD). OsARD1 is a metal-binding

protein that belongs to the cupin superfamily. Acireductone dioxygenases are unique proteins that can acquire

two different activities depending on the metal ion bound. Ectopically expressed apo-OsARD1 preferentially

binds Fe2þ and reconstituted Fe-OsARD1 catalyzed the formation of 2-keto-pentanoate and formate from the

model substrate 1,2-dihydroxy-3-ketopent-1-ene and dioxygen, indicating that OsARD1 is capable of catalyzing

the penultimate step in the methionine cycle. Two highly homologous ARD genes were identified in rice.

OsARD1 mRNA levels showed a rapid, early and transient increase upon submergence and after treatment with

ethylene-releasing compounds. The second gene from rice, OsARD2, is constitutively expressed. Accumulation

ofOsARD1 transcript was observed in the same internodal tissues, i.e. the meristem and elongation zone, which

were previously shown to synthesize ethylene. OsARD1 transcripts accumulated in the presence of cyclohex-

imide, an inhibitor of protein synthesis, indicating that OsARD1 is a primary ethylene response gene. Promoter

analysis suggests that immediate-early regulation ofOsARD1by ethylene may involve an EIN3-like transcription

factor.OsARD1 is induced by low levels of ethylene. We propose that early feedback activation of the methionine

cycle by low levels of ethylene ensures the high and continuous rates of ethylene synthesis required for long-

term ethylene-mediated submergence adaptation without depleting the tissue of AdoMet.

Keywords: acireductone dioxygenase, methionine cycle, ethylene, immediate-early response, Oryza sativa,

submergence.

Introduction

The plant hormone ethylene is known to regulate some

aspects of adaptation to hypoxic stress. Rice is a semi-

aquatic plant that is well adapted to periods of low oxygen

supply which commonly occur during waterlogging or

flooding. Deepwater rice varieties are particularly well

adapted to survive low oxygen stress during long-term

flooding. These plants have an improved aeration system

through internal and external gas spaces, they produce

adventitious roots at the nodes to replace the soil-borne

roots, and they have the ability to induce rapid internodal

elongation to keep part of their foliage above water (Kende

et al., 1998). The gaseous hormone ethylene is central to

the regulation of induced aerenchyma formation (Justin

and Armstrong, 1987; Webb and Armstrong, 1986),

adventitious root growth (Lorbiecke and Sauter, 1999;

Suge, 1985) and internodal elongation (Kende et al., 1998).

The concentration of ethylene within the plant increases in

response to submergence, due both to physical entrap-

ment (Jackson, 1985) and increased biosynthesis (Metraux

and Kende, 1983; Raskin and Kende, 1984).

718 ª 2005 The AuthorsJournal compilation ª 2005 Blackwell Publishing Ltd

The Plant Journal (2005) 44, 718–729 doi: 10.1111/j.1365-313X.2005.02564.x

Ethylene is synthesized from S-adenosyl-L-methionine

(AdoMet). Formation of the ethylene precursor 1-aminocy-

clopropane-1-carboxylic acid (ACC) from AdoMet by ACC

synthase (ACS) is considered as the rate-limiting step in

biosynthesis of ethylene. Activity of ACS is increased in

partially submergeddeepwater rice (Cohen andKende, 1987)

and expression of two ACS genes from rice, Os-ACS1

(Zarembinski and Theologis, 1997) andOs-ACS5 (Zhou et al.,

2001, 2002), was shown to be upregulated upon submer-

gence.

Synthesis of ACC from AdoMet releases 5¢-methylthioa-

denosine (MTA) as a by-product that is recycled to methi-

onine in a salvage pathway. The reduced sulfur group is

conserved and the hydrocarbon released as ethylene is

replenished from the ribose unit of ATP that activates

methionine to yield AdoMet. The methionine salvage path-

way, also known in plants as the Yang cycle (Miyazaki and

Yang, 1987; Wang et al., 1982; Yang and Hoffman, 1984),

exists not only in plants but also in bacteria and in animals

where MTA is released during synthesis of the polyamines

spermidine and spermine. In bacteria and in plants

(Figure 6), MTA is depurinated to 5-methylthioribose

(MTR) through the enzymatic activity of MTA nucleosidase.

5-Methylthioribose kinase catalyzes the subsequent phos-

phorylation of the C-1 hydroxyl group of the ribosemoiety of

MTR to yield 5-methylthioribose-1-phosphate which in turn

undergoes enzymatic isomerization and dehydration to

acireductone in a reaction that is catalyzed by E1 enolase/

phosphatase. In the methionine cycle, acireductone and

dioxygen react to produce 2-keto-4-methylthiobutyrate

(KMTB), the immediate precursor of methionine, and for-

mate. Acireductone dioxygenase (ARD), which catalyzes this

step, was characterized in detail from the bacterium Klebsi-

ella oxytoca, previously named Klebsiella pneumoniae (Dai

et al., 1999, 2001). Its structure was determined by NMR

methods (Pochapsky et al., 2002) but no plant homolog has

yet been described. In K. oxytoca ARD is a unique enzyme in

that the apo-protein acquires two different enzymatic activ-

ities depending on the metal ion bound in the active center.

Fe-ARD catalyzes the methionine cycle conversion of acire-

ductone to KMTB with formate being released as a by-

product, whereas Ni-ARD catalyzes an off-pathway resulting

in formation of methylthiopropionate, formate and carbon

monoxide (Figure 6; Dai et al., 1999). Both enzyme activities

were previously purified from Klebsiella.

Biochemical studies on enzymes of the methionine

salvage pathway in plants were performed more than

20 years ago, but only now are the corresponding genes

being identified; this in turn allows us to study regulation of

the methionine cycle at the molecular level (Sauter et al.,

2004). One aspect that we were particularly interested in was

to understand how synthesis of ethylene and salvage of

methionine are coordinated. In stored apples, ethylene was

shown to be synthesized for several months even though the

supply of methioninewas not sufficient to sustainmore than

a few hours of ethylene production, indicating that methi-

onine recycling was important for long-term synthesis of

ethylene (Baur and Yang, 1972). In our study we report that

in rice an ARD-encoding gene is upregulated by ethylene,

thus revealing a positive regulatory loop from ethylene to

the methionine cycle.

Results

OsARD1 is transiently induced in submerged deepwater rice

OsARD1 was identified in two independent screens for dif-

ferentially expressed genes in deepwater rice. First we per-

formed subtractive hybridization screening to identify genes

that were expressed at elevated levels in adventitious roots

of deepwater rice after 2 or 6 h of submergence. Secondly

we screened for genes that were differentially expressed in

the youngest internode of deepwater rice after treatment

with 10 mM ACC. The cDNAs encoded an open reading

frame of 199 amino acids which was originally termed sip2

for submergence-induced protein2 (AF050200). A homolo-

gous expressed sequence tag (EST) from rice was identified

in a database search, obtained from the National Institute of

Agrobiological Resources (Ibaraki, Japan), sequenced and

originally termed sip2A (AF068332). Based on sequence

similarities and activity of the encoded proteins as described

below we renamed the gene products as OsARD1 (sip2) and

OsARD2 (sip2A). OsARD2 encoded a protein of 198 amino

acids with 85% identity and 93% similarity to OsARD1.

Southern blot analysis supported the idea that rice posses-

ses two ARD genes (data not shown).

Expression of OsARD1 was strongly induced in adventi-

tious roots and in the youngest internode of partially

submerged deepwater rice plants (Figure 1a). The increase

in abundance of transcript was high in the intercalary

meristem and in the elongation zone of the internode. Little

induction of gene expression was observed in differentiated

cells. A detailed time-course analysis showed the highest

abundance of OsARD1 transcript after 2 h of submergence

(Figure 1b). Between 2 and 6 h of submergence transcripts

were rapidly degraded and returned to approximately the

same levels as in controls (Figure 1b). OsARD1 transcripts

appeared to have a short half-life, as was particularly

obvious in elongating cells (Figure 1b). By contrast expres-

sion of OsARD2 was constitutive in adventitious roots and in

the youngest internode (Figure 1a).

OsARD1 encodes an acireductone dioxygenase, an enzyme

of the methionine cycle

OsARD1 was putatively assigned as an ARD based on

sequence similarity to an ARD enzyme that was previously

described from K. oxytoca (Dai et al., 1999; Figure 2). Most

Methionine cycle regulation by ethylene 719

ª 2005 The AuthorsJournal compilation ª 2005 Blackwell Publishing Ltd, The Plant Journal, (2005), 44, 718–729

higher eukaryotes possess small ARD gene families. Euk-

aryotic ARD proteins are highly conserved with 67% simi-

larity between human sipL/HsARD and OsARD1 (Figure 2).

Acireductone dioxygenases belong to the cupin superfamily

which have a characteristic b-barrel fold (Dunwell et al.,

2004). They possess one cupin domain consisting of two

motifs with two b-strands each and an intermotif loop region

which also contains two b-strands. In the active site of the

ARD enzyme from Klebsiella His96, His98, Glu102 and

His140 were implicated in Ni2þ binding. Arg104 was dis-

cussed as a candidate base for deprotonation of acireduc-

tone and Phe92 and Phe142 were proposed to assist in

orienting the substrate properly (Pochapsky et al., 2002). All

of these residues were found to be strictly conserved

throughout prokaryotic, animal and plant ARD proteins

(Figure 2). Some ARD proteins, including OsARD1, contain

the canonical destruction box motif RxxLxx(x)N (Figure 2;

Genschik et al., 1998) which may indicate that not only

OsARD1 mRNA (Figure 1b) but also OsARD1 protein may be

subject to rapid degradation.

Since ARD proteins from plants have not been described

previously and overall homology to the bacterial enzyme is

low, we characterized the enzymatic activity of rice OsARD1

to ensure correct identification. Matrix-assisted laser de-

sorption ionization-time of flight (MALDI-TOF) electrospray

mass spectra revealed that ectopically expressed apo-

OsARD1 has an observed mass of 23 604.29 � 0.87 Da,

consistent with the expected value of 23 601 Da.

Bacterial ARD can bind either Fe2þ or Ni2þ as co-factor

(Dai et al., 1999). Fe-ARD catalyzes the methionine path-

way reaction whereas Ni-ARD catalyzes an off-pathway

reaction. We tested the possibility that the plant apo-

protein may have similar metal binding capacity and

resulting dual functions as described for bacterial and

human homologs. Removal of metal ions and reconstitu-

tion of holo-OsARD1 indicated that bacterially expressed

OsARD1 preferentially bound Fe2þ rather than Ni2þ indi-

cating that the on-pathway was favored.

Dynamic light scattering (DLS) results for reconstituted Fe-

OsARD1 indicated that holo-Fe (II)-OsARD1 is a stable trimer,

with a radius ranging from3.456 to 4.354 nm. Enzyme assays

were performed using a model substrate. Kinetic studies on

Fe-OsARD1 were carried out by varying the concentration of

either substrate with a fixed amount of the second substrate

(Dai et al., 2001). The acireductone substrate 1,2-dihydroxy-

3-ketopent-1-ene (DHKP) is a dethio analog of 5¢-methyl-

thioribose-1-phosphate that was generated in situ from

the precursor 1-phosphonooxy-2,2-dihydroxy-3-oxohex-

ane (PDOH) by the action of E1 enolase-phosphatase (Zhang

et al., 2004). The natural substrate is not synthetically

available. However, previous work has shown that for the

Klebsiella ARD enzyme PDOH showed the same product

distributions as the natural substrate in vivo.

Completion of the E1 enolase-phosphatase reaction was

monitored spectroscopically. Loss of all intermediate was

measured at 278 nm, and a stable product level (the ARD

substrate) was indicated by constant absorbance at

305 nm (Zhang et al., 2004). After the E1 enolase-phos-

phatase reaction was completed, the oxidation reactions

were initiated by the addition of appropriate amounts of

O2 gas and OsARD1 and were followed by measuring the

change in absorption at 305 nm. Double-reciprocal plots

of initial reaction velocity versus concentration were

prepared by varying, for one, the concentration of PDOH

at saturating oxygen which yielded a straight line along

the 1/substrate axis. A linear fit of the data gave

y ¼ 48.453x þ 1.8737 with R2 ¼ 0.9973. Varying the

concentration of O2 in the presence of 60 lM DHKP

yielded similar kinetics. A linear fit of data gave

y ¼ 2.0031x þ 1.8613 with R2 ¼ 0.9836.

The kinetic constants of Fe-OsARD1were derived from the

plots of intercepts and slopes. Of the two substrates, O2 had

a larger effect on initial rates than DHKP. The Michaelis

constant, Km, for DHKP as substrate was 25.9 lM with a

maximum reaction velocity, Vmax, of 26.7 lmol min)1. The

Km with O2 as the substrate was 1.1 mM with a similar Vmax

of 26.9 lmol min)1 (Table 1). No significant production of

(a)

(b)

Figure 1. Expression of OsARD1 but not of OsARD2 was induced by

submergence in deepwater rice plants.

(a) Gene-specific probes for OsARD1 and OsARD2 were successively hybrid-

ized to an RNA blot with 18 lg RNA per lane isolated from adventitious roots

at the third node and from the intercalary meristem, the elongation zone and

the differentiation zone of the youngest internode of deepwater rice plants

which were partially submerged for 0, 2, 6 or 18 h. Loading of the gel was

analyzed by hybridization to an HvPA42 probe.

(b) Transient expression of OsARD1 in the meristem and elongation zone of

deepwater rice plants was analyzed in a detailed time course between 0 and

6 h of partial submergence using 30 lg RNA per lane. Ethidium bromide

staining is shown as a control for RNA loading.

720 Margret Sauter et al.


CO by Fe-OsARD1 was observed, indicating that Fe-OsARD1

catalyzed the on-pathway oxidation in the methionine

salvage pathway only. Compared with Fe-ARD from Klebsi-

ella, Fe-OsARD1 had less activity, with kOsARD1cat

(11.7 sec)1) < kARDcat (2.6 · 102 sec)1).

Ni-OsARD1 produced CO and no KMTB analog, indicating

that it possesses off-pathway activity only. However, overall

enzyme activity was low with a Km of 44.0 lM for the model

substrate DHKP. Dynamic light scattering analysis indicated

that reconstituted Ni-OsARD1 exists as a polymer with a

mean radius of 127.1 nm and probably consisting of

multiple types of oligomers. Thus, unlike bacterial or human

ARD (Pochapsky et al., 2002), both metal forms of OsARD1

oligomerize.

Figure 2. Sequences of ARD homologs from different taxa with conserved structural features.

Protein sequences of ARD homologs from O. sativa (OsARD1; accession number AF050200), Arabidopsis thaliana (accession number AAC14490), Hordeum vulgare

(IDI1; accession number AB025597), Zea mays (translated from overlapping ESTs BQ619277 and BQ618945), Homo sapiens (sipL; accession number BAA91901),

Xenopus laevis (accession number AAH75403), Drosophila melanogaster (accession number AAN11908), Caenorhabditis elegans (hypothetical protein F42F12.4,

accession number T22103), Neurospora crassa (accession number EAA27424), Schizosaccharomyces pombe (accession number T40726), Saccharomyces

cerevisiae (hypothetical protein YMR009w, accession number Q03677), Bacillus subtilis (ykrZ; accession number B69864), Yersinia pseudotuberculosis (accession

number CAH20116) and K. oxytoca (ARD; accession number A59159) were aligned to indicate conserved sequences. The black bar indicates the cupin domain. Bold

capital letters underneath the sequences represent amino acids which are generally conserved in metal-binding cupins. Grey boxes above the sequences indicate

motifs 1 and 2 of the cupin domain which are separated by the loop region as indicated. Four of the eight b-strands are indicated in motifs 1 and 2 by open arrows.

The boxed residues in motif 2 indicate the destruction box motif RxxLxxxN.



OsARD1 gene expression is regulated by ethylene

In the internodes of deepwater rice, the submergence signal

that results in induction of growth is sequentially mediated

by ethylene and by gibberellic acid (Kende et al., 1998). In

order to find out which signal was ultimately responsible for

regulation of OsARD1 we treated stem sections containing

the youngest growth-responsive internode with the natural

ethylene precursor ACC. 1-Aminocyclopropane-1-carboxylic

acid is readily converted to ethylene in most plant tissues

due to the constitutive presence of ACC oxidase activity

(Yang and Hoffman, 1984). Application of ACC resulted in

strong induction of OsARD1 within 40 to 90 min (Figure 3a).

This result was consistent with identification of OsARD1 in a

screen for ACC-induced genes.

Ethephon is an ethylene-releasing chemical. Similar to

ACC, ethephon was effective in inducing OsARD1 gene

expression (Figure 3b). Transcript levels increased

distinctly within 1 h and reached a maximum at 2 h after

onset of ethephon treatment. After 2 h transcript abun-

dance declined, thus mimicking the transient expression

pattern observed in submerged plants (Figure 1a,b). The

observed lag phase of ethephon-induced accumulation of

OsARD1 transcript of 1 h or less was shorter than that

observed in submerged plants. A shorter lag phase for

induction of OsARD1 gene expression by ethylene is in

good agreement with the known signaling cascade in

which ethylene acts downstream of the submergence

signal. As was observed with submergence, treatment

with ethephon induced OsARD1 expression in the meri-

stem and in the elongation zone (data not shown).

Expression of OsARD2 on the other hand was not altered

by ethephon (Figure 4a) or by submergence treatment

(Figure 1a).

In a subsequent experiment, stem sections were treated

with either ethephon, gibberellic acid A3 (GA3), 2,5-norbor-

nadiene (NBD), an inhibitor of ethylene action (Bleecker

et al., 1987; Sisler and Yang, 1984), or with a combination of

ethephon and NBD (Figure 3c). Two controls were included,

one at time zero and a second one after 2.5 h of incubation,

the same time used for hormone treatments. A weak

induction of OsARD1 was observed in control stem sections,

possibly caused by excision of stem sections due to ethylene

released as a result of wounding. Induction of expression of

OsARD1 by ethephon was confirmed. 2,5-Norbornadiene

also induced OsARD1 gene expression. It has been noted

before that NBD by itself can mimic the action of ethylene,

but when combined with ethylene NBD acts as a competitive

inhibitor of ethylene perception (Bleecker et al., 1987). When

ethylene was supplied in the presence of NBD we observed

suppression of ethylene-dependent OsARD1 gene induc-

tion. Gibberellic acid A3 did not induce OsARD1 expression

(Figure 3c). A time-course experiment with GA3-treated

stem sections confirmed these results (data not shown).

(b)

(c)

(a)

Figure 3. Expression of OsARD1 is regulated by ethylene.

(a) Stem sections containing the youngest, growth-responsive internode of

deepwater rice plants were incubated for 40, 90 or 180 min with 10 mM ACC.

Control sections were incubated without ACC for 0 and for 90 min. Ribonu-

cleic acid was isolated from a 10 mm zone of the internode containing the

meristem and part of the elongation zone. Twentymicrograms of RNA in each

lane was hybridized to an OsARD1-specific probe. As a control for RNA

loading, ribosomal RNA was stained with ethidium bromide (EtBr).

(b) Stem sections were incubated with 150 lM ethephon for 1, 2, 3, 4, 5 or 6 h.

Untreated control stem sections were harvested at 0 and at 6 h (C6). Levels of

OsARD1 transcript were analyzed in a Northern blot with 20 lg RNA per lane

using a gene-specific probe. As a control for RNA loading, ribosomal RNAwas

stained with EtBr.

(c) Stem sections were incubated for 2.5 h with 150 lM ethephon (E), 50 ll l)1

norbornadiene (NBD), 50 lM gibberellic acid A3 (GA) or with ethephon and

norbornadiene (EþNBD). Two controls without addition of any of these

compounds were incubated for 0 h (C0) and 2.5 h (C2.5), respectively. Twenty

micrograms of RNA per lane were analyzed from the elongation zone with an

OsARD1-specific probe. As a control for RNA loading, ribosomal RNA was

stained with EtBr.

Table 1 Kinetic constants of Fe-OsARD1

Substrate Km kcat Vmax

DHKP 25.9 lM 11.7 sec)1 26.7 lmol min)1

O2 1.1 mM 11.8 sec)1 26.9 lmol min)1



We conclude from these results that ethylene is the hormo-

nal signal responsible for regulation of the OsARD1 gene in

the youngest internode of deepwater rice stems.

OsARD1 is an immediate-early ethylene response gene

Rapid transient gene expression is a characteristic of early-

induced genes. They can be induced in the absence of de

novo protein synthesis. In order to test whether OsARD1 was

an early response gene, we analyzed ethylene-induced gene

expression in the presence of cycloheximide, an inhibitor of

protein synthesis. In the absence of ethylene, treatment with

cycloheximide resulted in the accumulation of high levels of

OsARD1 transcripts (Figure 4a). Treatment with ethephon

caused induction of OsARD1 expression as shown earlier

(Figures 3b,c and 4a). Treatment with cycloheximide and

ethephon led to higher transcript levels than those observed

with ethephon alone (Figure 4a). Hyperinduction by cy-

cloheximide is commonly observed with early-induced

genes and has been explained by reduced transcript degra-

dation or lack of gene repression due to a short-lived protein

involved in either of these processes (Horvath and Chua,

1996; Suzuki et al., 1998).

Concentrations of cycloheximide between 0.002 and

2 lg ml)1 did not induce expression of OsARD1 but

20 lg ml)1 (71.1 lM) of cycloheximide or higher effectively

did (Figure 4b). These higher concentrations were shown

to efficiently inhibit protein synthesis in plants (Berberich

and Kusano, 1997; Monroy et al., 1993). Expression of

OsARD2 was unaffected by treatment with cycloheximide,

even at high concentrations, indicating a very low turn-

over rate of OsARD2 mRNA (Figure 4a). As a control for

cycloheximide activity, we analyzed the expression of the

pyruvate decarboxylase 2 (PDC2) gene. Levels of PDC2

mRNA were induced by ethephon and were repressed by

cycloheximide at a concentration of 20 lg ml)1 or more in

ethephon-treated stem sections. In the absence of ethe-

phon, PDC2 transcript levels declined with or without

cycloheximide treatment, indicating that cycloheximide

specifically inhibited ethylene-induced accumulation of

PDC2 mRNA (Figure 4a).

We next compared the concentration of ethephon that

was required to induce OsARD1 gene expression with the

concentration of ethephon required to induce internodal

elongation (Figure 5a,b). Expression of OsARD1 was ele-

vated at 1.5 lM ethephon and remained elevated up to

150 lM ethephon (Figure 5a). Induction of growth of the

internode was observed to a minor degree at 15 lM, and

high growth rates were achieved with 150 lM ethephon

(Figure 5b). Thus, the ethephon concentration required to

induce maximal OsARD1 expression was two orders of

magnitude lower than that required to elicit a maximal

growth response. With direct application of ethylene gas

to deepwater rice stem sections, Bleecker et al. (1987)

obtained maximal growth induction at 5–10 p.p.m. C2H4.

Given our observation that OsARD1 was induced at a 100-

fold lower ethylene concentration, the effective concen-

tration would be at around 0.1 p.p.m. C2H4. Levels of

ethylene in the internode were shown to rise from 0.08 to

0.14 p.p.m. within the first hour and to 0.2 p.p.m. within

2 h of submergence (Rose-John and Kende, 1985). The

rise in ethylene concentration that occurs within the first

2 h of submergence in deepwater rice would thus be

sufficient to account for induction of OsARD1 as observed

in submerged plants (Figure 1b).

(b)

(a)

Figure 4. Expression of OsARD1 but not of OsARD2 is induced in the

presence of cycloheximide.

(a) Stem sections were incubated with 20, 100 or 500 lg ml)1 cycloheximide

in the presence or absence of 150 lM ethephon. Cycloheximide was added

0.5 h before the addition of ethephon. After 3 h, RNA was isolated from the

meristematic region. Thirty micrograms of RNA per lane was successively

hybridized to gene-specific probes for OsARD1, OsARD2 and PDC2. As a

control for RNA loading, ribosomal RNA was stained with ethidium bromide

(EtBr).

(b) The concentration dependence of induction of OsARD1 by cycloheximide

was examined by treating stem sections with increasing concentrations of the

protein synthesis inhibitor as indicated. Ribonucleic acid was isolated after

3 h, separated and hybridized to an OsARD1-specific probe. The autoradio-

graph was scanned and the signal intensities were quantified. The signal of

the control (0 lg ml)1 cycloheximide) was arbitrarily set to 1 and all other

signals were calculated as multiples of this value.



Discussion

OsARD1 catalyzes formation of a-ketomethylthiobutyrate

in the methionine cycle

Acireductone dioxygenase enzymes are unique in that one

and the same polypeptide can acquire a different enzymatic

activity depending on the bound metal. Fe-ARD acts in the

methionine cycle to produce KMTB and the by-product for-

mate whereas Ni-ARD catalyzes an off-pathway reaction that

produces methylthiopropionic acid, formate and carbon

monoxide. This dual function was first described for

K. pneumoniae ARD (Dai et al., 1999). Even though both

enzyme activities were isolated from Klebsiella (Dai et al.,

1999) the physiological significance of the off-pathway

reaction is not understood. The resulting reaction product,

methylthiopropionic acid, is cytotoxic. In cassava (Manihot

spec) leaves that were infected by Xanthomonas campestris

methylthiopropionate was identified as a blight-inducing

toxin (Perraux et al., 1986). Methylthiopropionate is pro-

duced by phytopathogens and by soil micro-organisms for

which it was described as an antimicrobial substance that

also affects the growth of plant seedlings (Kim et al., 2003).

Whether or not methylthiopropionate is produced by plant

cells is not known. One could hypothesize that methylthio-

propionate may be employed by plants to induce cell death.

In that context it should be kept in mind that ethylene is a

major regulator of programmed cell death in plants. On the

other hand, Ni-ARD in plants may be an unwanted enzyme

variant and oligomerization may be a mechanism to reduce

Ni-ARD activity. Consistent with this argument is our

observation that reconstituted multimeric Ni-OsARD1 was

far less active than the reconstituted monomeric enzyme

from Klebsiella. Compared with monomeric Fe-ARD from

Klebsiella, trimeric Fe-OsARD1 also had less activity. Hence,

conversion of monomeric to multimeric forms could be a

general means of regulating ARD activity in plants. Future

work will have to clarify whether oligomerization occurs

in planta, possibly in a regulated process.

Ethylene controls OsARD1 gene expression possibly

through an EIN3-like transcription factor

Ethylene is synthesized from AdoMet via a specific two-step

pathway involving ACC as an intermediate. In submerged

deepwater rice increased evolution of ethylene is seen as a

result of increased expression and elevated activities of ACC

synthase and ACC oxidase (Cohen and Kende, 1987; Mekh-

edov and Kende, 1996; Van der Straeten et al., 2001;

Zarembinski and Theologis, 1997). Long-term flooding

requires prolonged ethylene production and hence a con-

tinued supply of the immediate ethylene precursor AdoMet

which can be provided through the methionine cycle

(Miyazaki and Yang, 1987).

We showed that upon submergence OsARD1 was induced

in nodal roots and in the growing region of the youngest

internode of deepwater rice, the same tissueswhich produce

elevated levels of ethylene (Cohen and Kende, 1987).

OsARD1 was induced by submergence, by ACC, the natural

immediate precursor of ethylene, and by ethephon, an

ethylene-releasing chemical. Since ethephon causes neither

depletion of AdoMet nor production of ACC, our results

suggest that ethylene itself is responsible for induction of

OsARD1 rather than the ethylene biosynthesis intermediates

AdoMet or ACC. In support of this conclusion, we found that

ethylene perception was required for OsARD1 induction.

Ethylene signaling occurs through a kinase cascade

(Bleecker, 1999; Bleecker and Kende, 2000; Johnson and

Ecker, 1998;Kieber, 1997;Ouakedet al., 2003)whichactivates

one ormoreEIN3/EIL (ethylene-induced3/ethylene induced3-

like) transcription factors (Chao et al., 1997). Dimers of EIN3

were shown to interact with the PERE (primary ethylene

response element) element that is present in the promoter of

ERF1 (ethylene response factor1). Ethylene response factor1

belongs to a group of transcription factors which bind to the

GCC box that is commonly found in promoters of stress-

related ethylene-response genes (Fujimoto et al., 2000;

Ohme-Takagi and Shinshi, 1995). The ERF genes are primary

(b)

(a)

Figure 5. Expression of OsARD1 is induced at lower ethylene concentrations

than internodal growth.

(a) Stem sections were incubated with ethephon at the concentrations

indicated. After 2.5 h, 20 lg RNA per lane was analyzed for OsARD1

expression. As a control for RNA loading, ribosomal RNA was stained with

ethidium bromide (EtBr).

(b) In a second experiment, stem sections were treated with ethephon at the

same concentrations and ethephon-induced growth of stem sections was

measured after 24 h (-r-). Average values (�standard error) were obtained

from three independent experiments with 8 to 16 stem sections per time

point.



ethylene response genes (Fujimoto et al., 2000; Solano et al.,

1998; Suzuki et al., 1998). Regulation by EIN3/EIL was shown

for ERF1 (Fujimoto et al., 2000; Solano et al., 1998).

At position )209 upstream of the start ATG of OsARD1

(sequenced from the BAC clone AC027658) we identified

the sequence AGATACGT that is identical to the consensus

PERE sequence AGA/GTT/AC

A/GT characterized from a

tobacco EIL (Kosugi and Ohashi, 2000). Two more PERE-like

sequences were found further upstream in the promoter of

OsARD1. OsARD1 was induced in the presence of cyclo-

heximide as were ERF1, -2, -3, -4 and -5 from Arabidopsis

(Fujimoto et al., 2000). Unlike the ERF genes, OsARD1

represents an immediate-early ethylene response gene that

does not code for a transcription factor. Conserved PERE

sequences were also found in promoter regions that are

required for ethylene responsiveness of the E4 and LEACO1

(ACC oxidase) genes from tomato and of the GST1 (gluta-

thione-S-transferase) gene from carnation (Blume and

Grierson, 1997; Itzhaki et al., 1994; Montgomery et al.,

1993).

Levels of EIN3/EIL mRNA in Arabidopsis were not altered

in response to ethylene (Chao et al., 1997). Instead, the

abundance of EIN3 was shown to be regulated at the protein

level through ubiquitin-mediated protein degradation

(Gagne et al., 2004). In accordance with the assumption that

OsARD1 was regulated through an EIN3-like transcription

factor we found that gene induction occurred in the absence

of de novo protein synthesis indicating that it required post-

translational events only.

OsARD1 is regulated by low levels of ethylene

The two classes of ethylene receptors were reported to

possess different biochemical properties (Johnson and

Ecker, 1998; Sisler, 1991) with a broad range of affinity

towards ethylene between 0.1 to 5 nM (Bleecker, 1997).

However, more recently published data showed that all

members of the ethylene receptor families from tomato

and from Arabidopsis displayed similar ethylene-binding

activity (O’Malley et al., 2005). Based on these recent data

induction of OsARD1 at low levels of ethylene and induc-

tion of growth by concentrations of ethylene up to 100-fold

higher cannot simply be explained in terms of binding to

receptor proteins with different affinities towards ethylene.

In etiolated Arabidopsis seedlings ethylene inhibits elon-

gation of hypocotyls. Using an ein3-1/eil1-1 double mutant it

was shown that the ethylene response occurred in two

phases. Long-term growth was inhibited by ethylene in an

EIN3/EIL1-dependent manner whereas short-term inhibition

of hypocotyl growth was not mediated by EIN3/EIL1 (Binder

et al., 2004). The short-term response was less sensitive to

treatment with 1-methylcyclopropene, an inhibitor of ethy-

lene perception, and it was shown to be induced by

approximately 500-fold lower levels of ethylene (Binder

et al., 2004). This is similar to our observation that OsARD1

expression was induced at approximately 100-fold lower

ethylene levels than internodal growth. Binder et al. (2004)

reported further that the amplitude of the short-term

response was dose-independent between 1 nl l)1 and

10 ll l)1 ethylene. Again we observed a similar phenom-

enon with OsARD1 gene expression which was elevated to

the same level between 1.5 and 150 lM ethephon. The

responsiveness of OsARD1 to low levels of ethylene may

ensure that OsARD1 is induced early on when rice plants

become submerged. If ARD activity was limiting for methi-

onine recycling, gene activation may ensure rapid and

continuous ethylene synthesis without depletion of the

AdoMet pool.

Forward and backward regulation between ethylene

biosynthesis and methionine recycling

By utilizing the methionine cycle during ethylene synthesis,

the plant removes an inhibitory by-product and at the same

time it recycles this by-product to the ethylene precursor

AdoMet. In each round of themethionine cycle, the dicarbon

moiety of ethylene is replenished from the ribose of the ATP

that activates methionine to yield AdoMet (Miyazaki and

Yang, 1987; Yung et al., 1982). Thus high rates of ethylene

synthesis can occur at low steady-state AdoMet levels and

are essentially fueled by ATP.

It has long been recognized that ethylene synthesis is

highly regulated (Kende, 1993; Yang and Hoffman, 1984).

In some tissues, ethylene has the capacity to stimulate its

own synthesis through a positive-feedback loop. Mostly

ACS, as the first enzyme in the ethylene-specific biosyn-

thesis pathway, was shown to be regulated, even though

in some instances, such as in deepwater rice, ACO was

induced as well (Bleecker and Kende, 2000; Mekhedov and

Kende, 1996). While many reports exist on upregulation of

ethylene biosynthetic enzymes, inhibition of ACS has also

been reported. One potent inhibitor is in fact MTA, which

means that ACS activity is subject to feedback inhibition

by its by-product (Figure 6; Hyodo and Tanaka, 1986).

2-Keto-4-methylthiobutyrate also inhibits the activity of

ACS, albeit to a lesser degree (Hyodo and Tanaka, 1986;

Yu et al., 1979). The biochemical and/or molecular mech-

anisms of these regulatory loops are as of yet not

understood.

The fact that MTA can effectively suppress ethylene

synthesis necessitates its efficient metabolism when

ethylene is produced at high levels. Methylthioadenosine

nucleosidase, the first enzyme in themethionine cycle, has a

high affinity towards its substrate MTA which was shown to

be rapidly hydrolyzed in apple tissue (Adams and Yang,

1977; Guranowski et al., 1981). A similar conclusion was

drawn for KMTB metabolism in the mungbean (Miyazaki

and Yang, 1987).



Our report showed regulation of OsARD1 as an immedi-

ate-early response to ethylene. OsARD may be crucial and

thus subject to tight regulation because it regulates a branch

point in the methionine cycle. While the on-pathway results

in regeneration of methionine, the off-pathway produces a

cytotoxic compound. Reconstituted OsARD1 displayed a

much higher affinity towards Fe2þ, thus favoring themethio-

nine cycle reaction. Whether metal binding to apo-ARD is

a controlled process in plant cells still remains to be

established.

Experimental procedures

Plant material

Seeds of Oryza sativa L., indica cultivar Pin Gaew 56, were originallyobtained from the International Rice Research Institute, Los Banos,Philippines. Rice plants were grown as described (Sauter, 1997).Plants were partially submerged in a 600-l plastic tank filled with tapwater as described leaving approximately 30 cm of the leaf tipsabove the water surface (Lorbiecke and Sauter, 1999). Stem sectionscontaining the youngest growth-responsive internode were excisedas described by Raskin and Kende (1984) and treated eitherwith 50 lM GA3, 10 mM ACC, 50 ll l)1 norbornadiene, ethephon,cycloheximide or combinations of these compounds at the con-centrations and for the times indicated. Growth of the stem sectionswas determined with a ruler. After incubation, the meristematictissue was isolated from 0 to 5 mm above the second highest node.Tissue from the elongating zone was harvested between 5 and15 mmabove the second highest node and differentiated tissue washarvested from the oldest portion of the internode just below theyoungest node. Tissue was frozen in liquid nitrogen and stored at)70�C until use.

Subtractive hybridization and cDNA library screening

Polymerase chain reaction-based subtractive hybridizations wereperformed according to Buchanan-Wollaston and Ainsworth (1997)

as described (Lorbiecke and Sauter, 2002). To obtain a full-lengthcDNA of OsARD1, a k-ZAPII-cDNA library from deepwater rice wasscreened (Sauter et al., 1995) using a 373-bp cDNA fragment ofOsARD1. The probe was labeled with digoxigenin according to the‘DIG System User’s Guide’ (Boehringer, Mannheim, Germany). Sixstrongly hybridizing plaques were recovered from a total of 2 · 105

recombinant phages. The clone containing the longest insert wassequenced from both sides. The partial clone isolated in the sub-tractive hybridization was identical to nucleotides 496 to 867 of thefull-length cDNA. The sequence of the full-length clone was initiallytermed sip2 for submergence-induced protein 2 and was depositedin the GenBank database (AF050200). The gene has now beenrenamed OsARD1.

A database search identified two ESTs with similar sequences tosip2/OsARD1 (D48790 and D47064). Both were obtained from theRice Genome Research Program at the National Institute of Agro-biological Resources (Tsukuba, Ibaraki, Japan) and were fullysequenced. They differed only in the length of their 3¢ ends,probably resulting from different polyadenylation. The fully se-quenced EST D48790 was initially termed sip2A and was laterrenamed OsARD2. The sip2A/OsARD2 sequence was depositedunder the accession number AF068332. Upon request, all novelmaterials described in this publication will be made available in atimely manner for non-commercial research purposes.

Sequence analysis

DNA and protein homologs of ARD sequences were searched in theavailable protein and nucleic acid databases with the BLAST algo-rithm (Altschul et al., 1997). The sequence alignment was calculatedusing CLUSTALW (Thompson et al., 1994) and manually edited withBioEdit (Hall, 1999).

RNA blot analysis

RNA was isolated and hybridizations were carried out as described(Sauter, 1997). For Northern blot analysis 15–30 lg of total RNA wasused. Ribosomal RNA was stained with ethidium bromide and usedas loading control. Alternatively, the blots were hybridized to anHvPA42 probe as a control for RNA loading (Sauter, 1997). ForOsARD1, a 371-bp fragment isolated through differential screeningwas used as probe. This fragment corresponded to nucleotides 496to 867 of OsARD1 and included the 3¢ untranslated region (UTR) and123 nucleotides of the coding region. For OsARD2, a 467-bp frag-ment including the 3¢UTR and 219 bp of the coding region was usedas a probe. For PDC2, a fragment from the coding region corres-ponding to nucleotides 1260 to 1654 of the cDNA sequence(accession number U27350) was used.

Expression and purification of OsARD1

For enzyme studies the open reading frame of the OsARD1 cDNAwas inserted at the initiator methionine codon of pET3a (Novagen,Madison, WI, USA). Escherichia coli strain BL21, transformed withpET3a–OsARD1, was grown in Luria–Bertani (LB) medium contain-ing 50 mg l)1 ampicillin at 25�C. After addition of isopropyl-b-D-thiogalactoside (IPTG) for promoter induction cells were grown foran additional 12 h at 25�C before they were harvested by centrifu-gation, and stored frozen at )80�C.

All steps for OsARD1 purification were done at 4�C. Initially, wefollowed the published purification scheme for purifying ARD andARD’ (E2 and E2¢) from K. pneumoniae (Dai et al., 1999). However,we noted that much of the expressed protein was denatured, and

Figure 6. A model indicating the regulatory loops between ethylene synthe-

sis and the methionine cycle.

5¢-Methylthioadenosine (MTA) is formed as a by-product of ethylene biosyn-

thesis. It is a strong inhibitor of ethylene and polyamine synthesis. 5¢-Methylthioadenosine inhibits ACC synthase (ACS), the first enzyme commit-

ted to ethylene synthesis. The product released by Fe-ARD, KMTB, also

inhibits ACS. In rice, ethylene feedback activates OsARD1 as an immediate-

early response. ACO ¼ ACC oxidase.



the small amount of folded OsARD1 contained bound Fe, asdetermined by atomic absorption analysis. We therefore developeda purification procedure based on that used for purifying protoca-techuate 2,3-dioxygenase from Bacillus macerans (Wolgel andLipscomb, 1990). Frozen cell paste was thawed in four volumes(w/v) of buffer A [50 mM 3-(N-morpholino)-propanesulfonic acid(MOPS) buffer, pH 6.9, containing 100 lM ferrous ammoniumsulfate and 2 mM cysteine]. Both the iron salt and cysteine used toprepare buffer A were from freshly prepared 100· stock solutions.The frozen cells were sonicated for 3 · 35 sec with a FisherScientific sonic dismembrator model 300 (Pittsburgh, PA, USA) at75% of maximal output with 60-sec recovery intervals. Two milli-grams each of ribonuclease, deoxyribonuclease and 5.7 mg tolu-enesulfonyl chloride were then added. After stirring for 30 min, thecellular homogenate was centrifuged at 30 000 g for 30 min, and theprecipitate discarded. The supernatant was loaded onto a diethyl-aminoethyl (DEAE)-cellulose column (2.5 · 20 cm) equilibrated bybuffer A. The column was washed with 100 ml of buffer A, and alinear gradient (200 and 200 ml) from 0 to 0.5 M NaCl in buffer A wasapplied. Holo-OsARD1 eluted at approximately 0.2 M NaCl, asdetermined by UV–visible spectroscopy and gel electrophoresis.The fractions containing OsARD1 were concentrated, made 4 M inNaCl and applied to a Phenyl-Sepharose (Amersham Biosciences,Piscataway, NJ, USA) column (2.5 · 20 cm) that had been pre-equilibrated with 4 M NaCl in buffer A. The enzyme was eluted nearthe middle of a linear gradient decreasing from 4 to 0 M NaCl inbuffer A.

Removal and replacement of the metal ion in OsARD1

Apo-OsARD1 was prepared by dialysis of enzyme against 100 mM

EDTA in 20 mM Tris-HCl, pH 7.4. Fe2þ-OsARD1 could also bereconstituted by denaturing the sonicate in 8 M urea in 50 mM Tris-HCl, pH 7.4 and then renaturing by eightfold dilution with 50 mM

Tris-HCl, pH 7.4, followed by the two-column chromatographydescribed above. Ten milligrams of dimethylglyoxime (to removetrace amounts of Ni(II)) and 170 mg of FeSO4Æ7H2O were added forabout 4 g cell paste in the reconstitution of Fe2þ-OsARD1. Toreconstitute Ni2þ-OsARD1 the protein was unfolded with 8 M ureaand refolded in the presence of 20 mMNi2þ prior to purification on adesalting column.

Mass spectrometry, metal analyses and dynamic light

scattering characterization of OsARD1

Electrospraymass spectra were obtained on an Applied BiosystemsVoyager 2102 MALDI-TOF system (Framingham, MA, USA)operating in positive ion mode. The relative metal content of theenzyme was obtained by atomic absorption analysis. Dynamic lightscattering analyses were performed with 0.2 mM OsARD1 in 20 mM

Tris-HCl, pH 7.4, using a DynaPro-99 DLS apparatus (Protein Solu-tions Inc., Charlottesville, VA, USA).

Enzyme activity assays on OsARD1

A coupled a-keto acid assay was performed according to Dai et al.(2001) using a model substrate. To our knowledge the natural sub-strate has not been synthesized to date and is therefore unavailable.The immediate ARD model substrate DHKP is not stable to air forextended periods. Therefore it was synthesized in situ from PDOHprior to each assay using E1 enolase-phosphatase (Zhang et al.,2004). The formation of a-ketopentanoic acid (the product of the

on-pathway oxidation reaction catalyzed by Fe-ARD) by Fe-OsARD1was assayed by reduction of the keto acid with NADH. Using exactlythe same conditions it was shown previously for the Klebsiella ARDenzyme that PDOH showed the same product distributions as thenatural substrate in vivo (Dai et al., 2001).

Five microliters of E1 enzyme was added to an assay solutionmade from 980 ll of reaction buffer, 10 ll 1 mg ml)1 catalasesolution and 5 ll of E1 enolase-phosphatase substrate PDOH (Zhanget al., 2004) in a 1 ml septum-sealed cuvette. Upon completeconsumption of PDOH, as monitored by no further increase inabsorbance at 305 nm, the appropriate amount of O2 gas was addedto the reaction cuvette, along with 5 ll of OsARD1. The solution wasmixed by inversion and the progress of the reaction monitoredspectrophotometrically. When it was desired to monitor keto acidformation, 5 ll of 20 mM NADH was added after complete loss ofARD substrate (no absorbance at 305 nm) and 60 units of bovineheart lactate dehydrogenase (Sigma, St Louis,MO,USA)was added.Consumption of NADHwasmonitored by the change in absorbanceat 340 nm. The reactions were allowed to go to completion.

The CO assay was performed according to Sundin and Larsson(2002). Carbon monoxide formation was quantified by gas chroma-tography (GC). The GC system used was a Hewlett-Packard 6890equipped with an Agilent G2747A nickel catalyst system with aflame ionization detector. The injector temperature was 25�C, theoven temperature 60�C, the nickel catalyst temperature 375�C andthe detector temperature 250�C. The solution to be assayed wasplaced in a sealed vial and 1 ml CO liberating solution (7.5 g saponinin 1 M sulfuric acid) was injected into the vial. After vortexing for1 min, the sealed vial was shaken for 40 min at 250 rpm at 37�C. Analiquot (100 ll) of the gas phase was injected with a gas-tightsyringe onto the GC column. Ni-ARD from K. pneumoniae, whichproduces CO as a product of acireductone oxidation, was used as apositive control.

Kinetic studies were carried out by varying the concentration ofone substrate in a standard ARD assay (Dai et al., 2001) with a fixedamount of the second substrate. For the substrate DHKP, double-reciprocal plots, prepared by varying the concentration of substrateprecursor PDOH at saturating oxygen, gave a straight line along the1/substrate axis. For O2, similar kinetics was observed. The kineticconstants of OsARD1 were derived from the plots of intercepts andslopes. The background reaction rates were measured usinginjections of the buffer used to stabilize Fe-OsARD1 without enzymeunder the same conditions as the reaction.

Acknowledgements

We thank Dr T. Sasaki from the Japanese Rice Genome ResearchProgram of the National Institute of Agrobiological Resources(Ibaraki, Japan) for providing ESTs D40831 and D47064 corres-ponding to OsARD2. TCP thanks Michael Maroney (University ofMassachusetts) for helpful discussions regarding the purification ofFe-OsARD1. Support from NIH grant R01-GM067786 to TCP andfrom DFG grants SA495/7-1 and SA495/7-2 to MS is gratefullyacknowledged.

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Sequence information: The sequences described in this paper were deposited in the database under the accession numbers

AF050200 (OsARD1) and AF068332 (OsARD2).



The immediate-early ethylene response gene OsARD1 encodes an acireductone dioxygenase involved in...

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