Interference with the citrulline-based nitric oxide synthase
assay by argininosuccinate lyase activity in Arabidopsis
extracts
Rudolf Tischner
1,
*, Mary Galli
2,
*, Yair M. Heimer
3,
*, Sarah Bielefeld
1
, Mamoru Okamoto
2
,
Alyson Mack
2
and Nigel M. Crawford
2
1 Albrecht von Haller Institut fur Pflanzenwissenschaften, University of Gottingen, Germany
2 Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA, USA
3 Department of Dryland Biotechnologies, J. Blaustein Institute for Desert Research, Ben-Gurion University, Sede-Boker, Israel
Nitric oxide (NO) serves as a central signal in a wide
variety of processes, including vasodilation, neural
communication and immune function in animals [1],
and defense responses, hormonal signaling and flower-
ing in plants [2–6]. The primary mechanism for NO
synthesis in animals involves oxidation of l-arginine to
l-citrulline and NO, and requires NADPH and oxygen
[7–9]. This reaction is catalyzed by nitric oxide syn-
thase (NOS) enzymes, which require tetrahydrobiopter-
in (BH

Biological Sciences, University of California
at San Diego, La Jolla, CA 92093-0116, USA
Fax ⁄ Tel: +1 858 534 1637
E-mail:
*These authors contributed equally to this
work
(Received 23 February 2007, revised
24 May 2007, accepted 20 June 2007)
doi:10.1111/j.1742-4658.2007.05950.x
There are many reports of an arginine-dependent nitric oxide synthase
activity in plants; however, the gene(s) or protein(s) responsible for this
activity have yet to be convincingly identified. To measure nitric oxide syn-
thase activity, many studies have relied on a citrulline-based assay that
measures the formation of l-citrulline from l-arginine using ion exchange
chromatography. In this article, we report that when such assays are used
with protein extracts from Arabidopsis, an arginine-dependent activity was
observed, but it produced a product other than citrulline. TLC analysis
identified the product as argininosuccinate. The reaction was stimulated by
fumarate (> 500 lm), implicating the urea cycle enzyme argininosuccinate
lyase (EC 4.3.2.1), which reversibly converts arginine and fumarate to argi-
ninosuccinate. These results indicate that caution is needed when using
standard citrulline-based assays to measure nitric oxide synthase activity in
plant extracts, and highlight the importance of verifying the identity of the
product as citrulline.
Abbreviations
ADF, Arabidopsis-derived factor; ASL, argininosuccinate lyase; BH
4
, tetrahydrobiopterin; CaM, calmodulin; NO, nitric oxide; NOS, nitric oxide
synthase.
4238 FEBS Journal 274 (2007) 4238–4245 ª 2007 The Authors Journal compilation ª 2007 FEBS

in plants. The most recent attempt, which identified
the gene AtNOS1 [42], has subsequently been chal-
lenged [48–50], leading to the proposal that the gene
be renamed AtNOA1 for nitric oxide-associated [48].
Thus, a renewed effort was made to determine the
source of arginine-dependent NOS activity in plants,
using crude protein extracts from Arabidopsis leaves.
By employing the citrulline-based NOS assay, an argi-
nine-dependent activity was discovered that was
strongly stimulated by an extract of low molecular
weight compounds from Arabidopsis leaves and pro-
duced argininosuccinate rather than citrulline. These
results identify a reaction that is catalyzed by an activ-
ity unrelated to NOS and that can interfere with or
mask authentic NOS activity.
Results and Discussion
As a first approach to search for NOS activity in Arabid-
opsis, the citrulline-based NOS assay was used to test
extracts from Arabidopsis leaves. Crude protein extracts
(supernatant from a 2 · 10
4
g centrifugation) were incu-
bated with [
14
C]arginine, NADPH and mammalian
NOS cofactors (BH
4
, FMN, FAD, Ca
2+
and CaM). At

18000
delta cpm mg protein
-1
h
-1
12345
Fig. 1. Detection of arginine-dependent activity in Arabidopsis
extracts. Reactions measured the conversion of [
14
C]arginine to a
product that did not bind a cation exchange resin. The data are pre-
sented as delta c.p.m.Æmg
)1
proteinÆh
)1
, which refers to the c.p.m.
value of the test reaction minus the c.p.m. value from the control
reaction (reaction terminated immediately after the addition of
[
14
C]arginine). The average c.p.m. for the control reaction was
approximately 1800. Reactions were performed using the com-
plete, initial buffer containing NOS cofactors as described in Experi-
mental procedures. Reactions also contained the following
components: lane 1, crude protein extract from Arabidopsis leaves;
lane 2, desalted protein extract; lane 3, desalted protein extract
plus low molecular weight fraction (ADF); lane 4, same as lane 3
except that the desalted protein extract was boiled before the
assay; lane 5, same as lane 3 except that the ADF was boiled
before the assay. Data are averages from 10 reactions; error bars

dures). We performed an additional experiment to test
for flavin-dependent activity, using diphenylene iodoni-
um (an inhibitor of flavoproteins including animal
NOS), and found no inhibition of the activity at con-
centrations of diphenylene iodonium up to 10 lm (data
not shown). Second, the products of the reaction were
analyzed by one-dimensional TLC followed by auto-
radiography. No citrulline was detected on the auto-
radiograms; instead, an unidentified compound was
observed as the major reaction product (Fig. 3B).
Together, these results showed that the reaction had
no requirement for known NOS cofactors and did not
produce the NOS coproduct citrulline, indicating that
it was not a typical NOS reaction.
To identify the unknown compound, the reaction
products were analyzed by two-dimensional TLC on
silica gel plates.
14
C-Labeled argininosuccinate was the
only radiolabeled product identified (Fig. 4). No radio-
labeled products comigrating with citrulline, ornithine,
urea, valine, hydroxyarginine, agmatine, spermine,
spermidine, putrescine or proline were detected (Fig. 4
and data not shown).
0 5 10 15 20
0
2000
4000
6000
8000

The TLC plate was developed with acetonitrile ⁄ ammonium hydrox-
ide ⁄ water (4 : 1 : 1) and then autoradiographed.
Argininosuccinate lyase and NOS assay R. Tischner et al.
4240 FEBS Journal 274 (2007) 4238–4245 ª 2007 The Authors Journal compilation ª 2007 FEBS
Argininosuccinate is the immediate precursor to
arginine in the urea cycle, and is converted to arginine
and fumarate by argininosuccinate lyase (ASL;
EC 4.3.2.1; Fig. 5). Argininosuccinate is normally
made from citrulline and aspartate by argininosuccin-
ate synthetase, but it can also be produced by ASL in
a reverse reaction. ASL is found in plants, animals and
bacteria, and requires no external cofactors or metal
ions for catalytic activity [51]. The forward reaction
(argininosuccinate to arginine and fumarate) is
favored; reported K
m
values for argininosuccinate
range from 0.13 mm in jack bean [52] to 0.2 mm in
human liver [53], whereas the reported K
m
values for
the reverse reaction are 5.3 mm for fumarate and
3.0 mm for arginine [53].
If argininosuccinate synthesis is being catalyzed by
ASL in the Arabidopsis protein extracts, then fumarate
would be needed as a cosubstrate, and fumarate would
be the active component in the ADF preparation.
Therefore, partially purified ADF was treated with
fumarase, which converts fumarate to malate. After
fumarase treatment, ADF no longer enhanced the pro-

strates and products for ASL.
R. Tischner et al. Argininosuccinate lyase and NOS assay
FEBS Journal 274 (2007) 4238–4245 ª 2007 The Authors Journal compilation ª 2007 FEBS 4241
instead of fumarate, no activity was detected (data not
shown). When the amount of product produced was
measured as a function of fumarate concentration
using desalted Arabidopsis extracts, the data showed a
saturation curve (Fig. 7), which yielded a K
m
(fuma-
rate) of 4.5 mm, similar to what is reported for human
liver [53]. The reaction could be strongly inhibited (by
97%) by 0.3 mm argininosuccinate (data not shown),
the substrate for the favored forward reaction. Desalted
protein extracts from Escherichia coli were also tested,
and the same argininosuccinate product was produced
with ADF or fumarate (Fig. 6).
Our results show that when the citrulline-based
assay is employed, protein extracts from Arabidopsis
catalyze a reaction with arginine that mimics an NOS
reaction. This reaction, however, produces argininosuc-
cinate, not citrulline, and requires fumarate, indicating
that ASL is catalyzing the reaction. Because arginino-
succinate does not bind the cation exchange column,
the signal from the reaction could be mistaken for
NOS activity. Initially, it was puzzling why activity
was obtained in crude Arabidopsis extracts without
added fumarate (ADF); however, several articles have
reported that fumarate levels can be quite high in
plants, especially in Arabidopsis, where it is reported to

with SDs.
Treatment Crude extract Desalted extract Desalted extract + ADF Desalted extract + treated ADF
Activity 16 354 ± 1267 1762 ± 119 16 085 ± 1440 2583 ± 183
Fig. 6. Fumarate can replace ADF as a cosubstrate for the reaction.
Desalted protein extracts from Arabidopsis leaves or from E. coli
pellets were incubated with [
14
C]arginine in 50 m M NaPO
4
with or
without partially purified ADF (37 lg) or fumarate (final concentra-
tion of 12.5 m
M) as indicated. The reaction products were treated
with cation exchange resin, and unbound material was spotted
onto a silica TLC plate as described. The one-dimensional TLC was
developed with acetonitrile ⁄ ammonium hydroxide ⁄ water (4 : 1 : 1)
and then autoradiographed.
Fig. 7. The fumarate-dependent reaction follows Michaelis–Menten
kinetics. Reactions were performed with [
14
C]arginine (20 lM), de-
salted Arabidopsis protein extract and fumarate as described above.
The amount of product (shown as delta c.p.m.) was determined as
a function of fumarate concentration. The inset shows the double
reciprocal plot used to calculate K
m
.
Argininosuccinate lyase and NOS assay R. Tischner et al.
4242 FEBS Journal 274 (2007) 4238–4245 ª 2007 The Authors Journal compilation ª 2007 FEBS
4-(2-aminoethyl)-benzolsulfonylfluorid, 1 · Roche Protease

tions were determined using the Bradford Assay (Biorad,
Hercules, CA, USA).
ADF preparation
Leaf tissue (50 g) from 3-week-old Arabidopsis plants was
boiled for 15 min in 100 mL of water containing 1 mm
b-mercaptoethanol. The boiled extract was centrifuged at
2 · 10
4
g at room temperature (Beckman J2-HS, rotor
JA-20), and the supernatant was lyophilized. Resuspended
material was used directly or partially purified on a
72 cm · 1.5 cm column containing G-15 Sephadex (Sigma,
St Louis, MO, USA) in water. Fractions were assayed for
activation of desalted protein extracts. Active fractions were
subsequently pooled and applied to a Q-Separose FF
column (Amersham) equilibrated with 50 mm NaPO
4
(pH 7.4). The column was eluted with increasing concentra-
tions of NaCl. Active fractions eluted between 0.4 m and
0.5 m NaCl. These fractions were pooled, lyophilized, and
separated on the same G-15 Sephadex column as described
previously. Fractions were assayed for activation potential,
combined, lyophilized, and resuspended into 100 lLof
water.
Enzyme assays and cation exchange
chromatography
Thirty to 150 lg of protein extract (either desalted or
crude) was used for each assay. The initial assay buffer
with NOS cofactors contained 1 mm NADPH, 130 lm
BH

supernatant was evaporated to dryness in a speedvac, and
resuspended in 10% of the original volume with 10% aceto-
nitrile in water. For one-dimensional TLC, 1 lL was spot-
ted on silica gel TLC plates (Whatman #4420221, Clifton,
NJ, USA) and developed with acetonitrile ⁄ water ⁄ ammo-
nium hydroxide 4 : 1 : 1. For two-dimensional TLC, 4 lL
of this mixture was spotted on silica gel TLC plates
(Whatman #4420221) and developed with n-butanol ⁄
methanol ⁄ ammonium hydroxide ⁄ water (33 : 33 : 24 : 10) in
the first dimension. After drying, the plates were developed
in the second dimension with chloroform ⁄ methanol ⁄ acetic
acid (2 : 4 : 4). Standards of known amines and amino acids
were run in parallel; they were spotted with the radioactive
material and detected by spraying with ninhydrin. Radioac-
tive arginine derivatives were detected directly on the TLC
plates by autoradiography (Hyblot CL, Denville Scientific,
Metuchen, NJ, USA).
Acknowledgements
We thank Dr Fujinori Hanawa for his excellent techni-
cal advice. This work was funded by grant from the
National Institutes of Health (GM40672).
References
1 Ignarro LJ (2000) Nitric Oxide, Biology and Pathobiology.
Academic Press, San Diego, CA.
2 Lamattina L, Garcia-Mata C, Graziano M & Pagnussat G
(2003) Nitric oxide: the versatility of an extensive signal
molecule. Annu Rev Plant Biol 54, 109–136.
3 Neill SJ, Desikan R & Hancock JT (2003) Nitric oxide
signalling in plants. New Phytologist 159, 11–35.
R. Tischner et al. Argininosuccinate lyase and NOS assay

like protein from Bacillus subtilis. J Biol Chem 277,
16167–16171.
14 Adak S, Bilwes AM, Panda K, Hosfield D, Aulak KS,
McDonald JF, Tainer JA, Getzoff ED, Crane BR &
Stuehr DJ (2002) Cloning, expression, and characteriza-
tion of a nitric oxide synthase protein from Deinococcus
radiodurans. Proc Natl Acad Sci USA 99 , 107–112.
15 Kers JA, Wach MJ, Krasnoff SB, Widom J, Cameron
KD, Bukhalid RA, Gibson DM, Crane BR & Loria R
(2004) Nitration of a peptide phytotoxin by bacterial
nitric oxide synthase. Nature 429, 79–82.
16 Wang ZQ, Lawson RJ, Buddha MR, Wei CC, Crane BR,
Munro AW & Stuehr DJ (2007) Bacterial flavodoxins
support nitric oxide production by Bacillus subtilis nitric-
oxide synthase. J Biol Chem 282, 2196–2202.
17 Theologis A, Ecker JR, Palm CJ, Federspiel NA, Kaul
S, White O, Alonso J, Altafi H, Araujo R, Bowman CL
et al. (2000) Analysis of the genome sequence of the
flowering plant Arabidopsis thaliana. Nature 408, 796–
815.
18 International Rice Genomic Sequencing Project (2005)
The map-based sequence of the rice genome. Nature
436, 793–800.
19 Klepper L (1979) Nitric-oxide (NO) and nitrogen-diox-
ide (NO2) emissions from herbicide-treated soybean
plants. Atmos Environ 13, 537–542.
20 Leshem Y & Haramaty E (1996) The characterization
and contrasting effects of the nitric oxide free radical in
vegetative stress and senescence of Pisum sativum Linn.
foliage. J Plant Physiol 148, 258–263.

1247–1258.
28 Yamasaki H, Sakihama Y & Takahashi S (1999) An
alternative pathway for nitric oxide production in
plants: new features of an old enzyme. Trends Plant Sci
4, 128–129.
29 Desikan R, Griffiths R, Hancock J & Neill S (2002) A
new role for an old enzyme: nitrate reductase-mediated
nitric oxide generation is required for abscisic acid-
induced stomatal closure in Arabidopsis thaliana. Proc
Natl Acad Sci USA 99, 16314–16318.
30 Meyer C, Lea US, Provan F, Kaiser WM & Lillo C
(2005) Is nitrate reductase a major player in the plant
NO (nitric oxide) game? Photosynth Res 83, 181–189.
31 Tischner R, Planchet E & Kaiser WM (2004) Mitochon-
drial electron transport as a source for nitric oxide in
the unicellular green alga Chlorella sorokiniana. FEBS
Lett 576, 151–155.
32 Planchet E, Jagadis Gupta K, Sonoda M & Kaiser WM
(2005) Nitric oxide emission from tobacco leaves and
cell suspensions: rate limiting factors and evidence for
the involvement of mitochondrial electron transport.
Plant J 41, 732–743.
Argininosuccinate lyase and NOS assay R. Tischner et al.
4244 FEBS Journal 274 (2007) 4238–4245 ª 2007 The Authors Journal compilation ª 2007 FEBS
33 Gupta KJ, Stoimenova M & Kaiser WM (2005) In
higher plants, only root mitochondria, but not leaf
mitochondria reduce nitrite to NO, in vitro and in situ.
J Exp Bot 56, 2601–2609.
34 Bethke PC, Badger MR & Jones RL (2004) Apoplastic
synthesis of nitric oxide by plant tissues. Plant Cell 16,

He SY (2006) Plant stomata function in innate immu-
nity against bacterial invasion. Cell 126, 969–980.
44 Bredt DS & Snyder SH (1989) Nitric oxide mediates
glutamate-linked enhancement of cGMP levels in the
cerebellum. Proc Natl Acad Sci USA 86, 9030–9033.
45 Tian QY, Sun DH, Zhao MG & Zhang WH (2007)
Inhibition of nitric oxide synthase (NOS) underlies alu-
minum-induced inhibition of root elongation in Hibiscus
moscheutos. New Phytol 174, 322–331.
46 Xu MJ, Dong JF & Zhu MY (2005) Nitric oxide medi-
ates the fungal elicitor-induced hypericin production of
Hypericum perforatum cell suspension cultures through
a jasmonic-acid-dependent signal pathway. Plant Physiol
139, 991–998.
47 Conrad KP, Powers RW, Davis AK & Novak J (2002)
Citrulline is not the major product using the standard
‘NOS activity’ assay on renal cortical homogenates. Am
J Physiol Regul Integr Comp Physiol 282, R303–R310.
48 Crawford NM, Galli M, Tischner R, Heimer YM,
Okamoto M, Mack A (2006) Response to Zemojtel
et al. Plant nitric oxide synthase: back to square one.
Trends Plant Sci 11, 526–527.
49 Guo FQ (2006) Response to Zemojtel et al. Plant nitric
oxide synthase: AtNOS1 is just the beginning. Trends
Plant Sci 11, 527–528.
50 Zemojtel T, Frohlich A, Palmieri MC, Kolanczyk M,
Mikula I, Wyrwicz LS, Wanker EE, Mundlos S, Ving-
ron M, Martasek P et al. (2006) Plant nitric oxide syn-
thase: a never-ending story? Trends Plant Sci 11, 524–
525.