Báo cáo khoa học: Interference with the citrulline-based nitric oxide synthase assay by argininosuccinate lyase activity in Arabidopsis extracts docx

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 4 ), FMN, FAD, calmodulin (CaM), and Ca 2+ . Three isoforms of highly conserved NOS enzymes have been identified in mammals: neuronal NOS (nNOS or NOS-I), inducible NOS (iNOS or NOS-II), and endothelial NOS (eNOS or NOS-III). NOS enzymes contain an N-terminal oxygenase domain and a C-terminal reductase domain connected by a CaM- binding hinge region. NOS enzymes are also found in specific species of fish, invertebrates, protozoa and fungi [10–12]. Even bacteria contain genes coding for truncated NOS proteins with homology to the oxygen- ase domain of mammalian NOS, and these enzymes have nitration or NO synthesis activity [13–16]. Despite the high degree of conservation found among NOS enzymes, no protein with significant sequence similarity has been identified in plants, including Arabidopsis [17] and rice [18], the genomes of which have been sequenced. Plants can produce and release significant amounts of NO, especially under hypoxic conditions or during infection [2,3,19–27]. One source of NO is nitrite, which can be converted to NO Keywords Arabidopsis; argininosuccinate lyase; citrulline; nitric oxide Correspondence N. M. Crawford, Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0116, USA Fax ⁄ Tel: +1 858 534 1637 E-mail: ncrawford@ucsd.edu *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 by: (a) plant nitrate reductase [22,28–30]; (b) mito- chondria [31–33]; and (c) nonenzymatic processes [34,35]. There is also ample evidence from biochemical and pharmacological data that an arginine-dependent mechanism analogous to animal NOS reactions exists in plants [26,36–43]; however, the identity of the argi- nine-dependent activity in plants has yet to be conclu- sively determined. Some of the evidence supporting an arginine-depen- dent mechanism in plants comes from commercially available ‘NOS assay kits’ (citrulline-based assays) that measure the conversion of arginine to citrulline using ion exchange chromatography [44]. Radiolabeled argi- nine is provided as a substrate, and is then separated from reaction products by cation exchange chromato- graphy. Positively charged arginine binds the ion exchange resin but citrulline does not. The unbound fraction, which is generally assumed to be citrulline, is measured in a scintillation counter. Examples of the use of this assay include the analysis of NOS activity in aluminum-treated Hibiscus [45], in pea peroxisomes [38], and in elicitor-treated Hypericum cells [46]. Although the assay is quick and sensitive, it does not identify the product as citrulline; any arginine deriva- tive that does not bind to the cation exchange resin will give a signal. The discovery of a product from a typical NOS reaction that is not citrulline was reported in a mammalian system [47]. There have been several attempts to identify the source responsible for arginine-dependent NOS activity 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 the end of the reaction, unreacted arginine was removed from the assay mixture with a cation exchange resin. Radioactive material that did not bind the resin, pre- sumably citrulline, was measured by scintillation count- ing. The signal obtained from a complete reaction (Fig. 1, lane 1) was up to 20 times higher than that from the control, which was a complete reaction terminated immediately after the addition of radiolabeled arginine. To determine potential cofactor requirements for the observed activity, leaf extracts were desalted using G-25 Sephadex to remove low molecular weight compounds. The low molecular weight compounds retained by the G-25 column were also collected by further elution of the column as described in Experimental procedures. Desalted protein extracts alone had greatly reduced levels of activity (Fig. 1, lane 2), indicating that a low molecular weight compound(s) from the extract was necessary for activity. Adding back the low molecular weight fraction from the G-25 column to the desalted protein extract restored activity (Fig. 1, lane 3). We named the low molecular weight fraction ADF, for 0 2000 4000 6000 8000 10000 12000 14000 16000 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 indicate SDs. R. Tischner et al. Argininosuccinate lyase and NOS assay FEBS Journal 274 (2007) 4238–4245 ª 2007 The Authors Journal compilation ª 2007 FEBS 4239 Arabidopsis-derived factor. The stimulation of activity by ADF was positively correlated with the amount of ADF added (Fig. 2). Boiled protein extract showed very little activity in the presence of ADF (Fig. 1, lane 4), whereas boiled ADF (Fig. 1, lane 5) stimulated activ- ity as much as untreated ADF (Fig. 1, lane 3) when added to the desalted extract, indicating that ADF is heat stable. These results suggested that an arginine-dependent activity was present in protein extracts of Arabidopsis leaves, and that a low molecular weight molecule(s) was required for this activity. To determine whether this activity was similar to that of mammalian NOS enzymes, two experiments were performed. First, cofactors essential for NOS activity (BH 4 , FMN, FAD, Ca 2+ and CaM) were omitted from the reac- tion. Robust activity was still observed for crude pro- tein extract and desalted protein extract to which ADF was added (Fig. 3A, lanes 1–4). For these reactions, partially purified ADF preparations were used (purifi- cation involved boiling leaf extracts and then passing them through two gel filtration columns and an anion exchange column, as described in Experimental proce- 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 10000 12000 14000 16000 delta cpm mg protein -1 h -1 µL ADF Fig. 2. Dependence of arginine-dependent reaction on ADF. Desalt- ed protein extracts were incubated with [ 14 C]arginine and increas- ing amounts of partially purified ADF, and then assayed for activity as described in Fig. 1. Data points are averages from five repli- cates; error bars indicate SDs. A B Fig. 3. The arginine-derived reaction product is not citrulline. Crude protein extracts (lanes 1 and 2), desalted protein extracts (lanes 3 and 4), boiled extracts (lanes 5 and 6) and no extract (lane 7) were assayed with [ 14 C]arginine and 50 mM NaPO 4 (i.e. with no NOS co- factors) in 50 lL as described in Experimental procedures. Partially purified ADF (37 lg) was included in lanes 2, 4 and 6. Reaction products (radioactive material that did not bind the cation exchange column) were analyzed by scintillation counting (A) and TLC (B). 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- duction of argininosuccinate (Table 1). Next, fumarate was tested as a replacement for ADF in the reactions. Desalted protein extracts from Arabidopsis were incu- bated with either ADF or fumarate; both reactions produced the same product, which comigrated with argininosuccinate by TLC analysis (Fig. 6). When maleic acid (the cis-isomer of fumarate) was used Origin 1-D 2-D Ornithine Argininosuccinate Hydroxyarginine Citrulline Fig. 4. Two-dimensional TLC analysis of reaction product. A reaction with [ 14 C]arginine, desalted protein extract and ADF was performed as described in Fig. 1, and then treated with cation exchange resin. A portion of the unbound material (5 lL out of a total of 100 lL) was spotted together with unlabeled markers onto a silica TLC plate. The TLC plate was developed with two solvent systems as follows: first dimension, n-butanol ⁄ methanol ⁄ ammo- nium hydroxide ⁄ water (33 : 33 : 24 : 10); second dimension, chloroform ⁄ methanol ⁄ acetic acid (2 : 4 : 4). The plate was then autoradiographed. The markers were visualized by ninhydrin stain- ing, and then marked as dashed lines on the autoradiogram. The origin was marked with an ink spot. Fig. 5. Urea cycle. (A) Schematic diagram of the urea cycle. (B) Structures of the sub- 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 be one of the most abundant organic acids [54,55]. The same activity can also be observed in protein extracts of E. coli, but only if fumarate or low mole- cular weight compounds from Arabidopsis leaves are added to the E. coli extracts. These results demonstrate the importance of verify- ing the identity of the products in standard citrulline- based NOS assays of plant and, especially, Arabidopsis extracts. Until such tests are performed, the results from such assays cannot be used to support the exis- tence of arginine-dependent NOS activity in plants. Experimental procedures Plant material and protein extractions Leaves from 3-week old Arabidopsis plants (ecotype Columbia) grown under 16 h light conditions were har- vested and ground in liquid N 2 with a mortar and pestle. Extraction buffer (2.5 mL of 50 mm Hepes, pH 7.4, 1 mm EDTA, 10 mm MgCl 2 ,1mm b-mercaptoethanol, 1 mm Table 1. Fumarase destroys ADF activity. [ 14 C]Arginine was incubated with desalted protein extracts from Arabidopsis leaves and partially purified ADF that was untreated or treated with fumarase as indicated. Treated ADF (500 lL) was incubated with 50 U of fumarase, and an aliquot was used in the assay after fumarase was inactivated by heat. Activity is presented as delta c.p.m.Æmg )1 proteinÆh )1 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 Inhibitor cocktail per gram fresh weight) was mixed with the ground plant material, and samples were centrifuged (2 · 10 4 g) for 10 min at 4 °C (Beckman J2-HS, rotor JA-20, Palo Alto, CA, USA). The supernatant (crude pro- tein extract) was either used directly or further desalted on a G-25 Sephadex gel filtration column (PD-10 column from GE Healthcare, Piscataway, NJ, USA), according to the manufacturer’s instructions. Briefly, 2.5 mL protein extract was applied to a PD10 column of 10 mL bed volume and then washed with extraction buffer. The first 2.5 mL of elu- ant was discarded, the next 3.5 mL (excluded volume) was collected (called desalted protein extract), and the next 3.5 mL (included volume) was collected and contained small molecules. Extracts were concentrated in a Centricon- 30 filter device (Millipore, Bedford, MA, USA) at 4 °C. For E. coli protein extracts, cell pellets were resuspended in lysis buffer (25 mm Hepes, 0.7 mm Na 2 HPO 4 , 137 m m NaCl, 5 mm KCl, pH 7.4), incubated on ice for 20 min with 1 mgÆmL )1 lysozyme, and sonicated. Lysate was cen- trifuged at 100 000 g for 1 h (Beckman ultracentrifuge L7, rotor SW51), desalted on a PD-10 column, and concen- trated with a Centricon-30 filter device. Protein concentra- 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 4 , 520 lm FMN, 200 lm FAD, 1 lm CaM, 1 mm CaCl 2 ,50mm Hepes (pH 7.4), and 10 lm [ 14 C]arginine (Amersham). Assays with desalted extracts were supplied with ADF (1–5 lL) unless indicated otherwise. Subse- quently, the initial assay buffer was replaced with 50 mm NaPO 4 buffer (pH 7.4) (i.e. with no NOS cofactors) and 10 lm [ 14 C]arginine. Reactions were incubated at 30 °C for 1 h, and terminated by boiling or immediately applying the reaction to spin columns (Corning, NY, USA) containing DOWEX 50WX8-400 (Sigma) cation exchange resin. DOWEX columns were prepared as previously described [56], and the flow-through was counted in a scintillation counter. TLC Following treatment with the cation exchange resin, 10% of the unbound material was counted in a scintillation counter and the remaining 90% was used for TLC analysis as fol- lows. The unbound material was washed with four volumes of cold acetonitrile and centrifuged for 10 min at 15 000 g (Eppendorf 5415C centrifuge; Brinkmann, Westbury, NY, USA) to precipitate large molecular weight compounds. The 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 FEBS Journal 274 (2007) 4238–4245 ª 2007 The Authors Journal compilation ª 2007 FEBS 4243 4 Wendehenne D, Durner J & Klessig DF (2004) Nitric oxide: a new player in plant signalling and defense responses. Curr Opin Plant Biol 7, 449–455. 5 Crawford NM & Guo FQ (2005) New insights into nitric oxide metabolism and regulatory functions. Trends Plant Sci 10, 195–200. 6 Delledonne M (2005) NO news is good news for plants. Curr Opin Plant Biol 8, 1–7. 7 Alderton WK, Cooper CE & Knowles RG (2001) Nitric oxide synthases: structure, function and inhibition. Biochem J 357, 593–615. 8 Stuehr DJ, Santolini J, Wang ZQ, Wei CC & Adak S (2004) Update on mechanism and catalytic regulation in the NO synthases. J Biol Chem 279, 36167–36170. 9 Li H & Poulos TL (2005) Structure–function studies on nitric oxide synthases. J Inorg Biochem 99, 293–305. 10 Ninnemann H & Maier J (1996) Indications for the occurrence of nitric oxide synthases in fungi and plants and the involvement in photoconidiation of Neurospora crassa. Photochem Photobiol 64, 393–398. 11 Golderer G, Werner ER, Leitner S, Grobner P & Werner- Felmayer G (2001) Nitric oxide synthase is induced in sporulation of Physarum polycephalum. Genes Dev 15, 1299–1309. 12 Torreilles J (2001) Nitric oxide: one of the more con- served and widespread signaling molecules. Front Biosci 6, D1161–D1172. 13 Adak S, Aulak KS & Stuehr DJ (2002) Direct evidence for nitrate oxide production by a nitric-oxide synthase- 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. 21 Wildt J, Kley D, Rockel A, Rockel P & Segschneider HJ (1997) Emission of NO from several higher plant species. J Geophys Res Atmospheres 102, 5919–5927. 22 Rockel P, Strube F, Rockel A, Wildt J & Kaiser WM (2002) Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro. J Exp Bot 53, 103–110. 23 Morot-Gaudry-Talarmain Y, Rockel P, Moureaux T, Quillere I, Leydecker MT, Kaiser WM & Morot-Gaudry JF (2002) Nitrite accumulation and nitric oxide emission in relation to cellular signaling in nitrite reductase antisense tobacco. Planta 215, 708–715. 24 Conrath U, Amoroso G, Kohle H & Sultemeyer DF (2004) Non-invasive online detection of nitric oxide from plants and some other organisms by mass spec- trometry. Plant J 38, 1015–1022. 25 Kaiser WM, Planchet E & Sonoda M (2004) NO emis- sion by plants, and sources for NO. Nitric Oxide Biol Chem 11, 37. 26 Zeidler D, Zahringer U, Gerber I, Dubery I, Hartung T, Bors W, Hutzler P & Durner J (2004) Innate immu- nity in Arabidopsis thaliana: lipopolysaccharides activate nitric oxide synthase (NOS) and induce defense genes. Proc Natl Acad Sci USA 101, 15811–15816. 27 Mur LA, Santosa IE, Laarhoven LJ, Holton NJ, Harren FJ & Smith AR (2005) Laser photoacoustic detection allows in planta detection of nitric oxide in tobacco following challenge with avirulent and virulent Pseudomonas syringae pathovars. Plant Physiol 138, 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, 332–341. 35 Bethke PC, Gubler F, Jacobsen JV & Jones RL (2004) Dormancy of Arabidopsis seeds and barley grains can be broken by nitric oxide. Planta 219, 847–855. 36 Delledonne M, Xia Y, Dixon RA & Lamb C (1998) Nitric oxide functions as a signal in plant disease resis- tance. Nature 394, 585–588. 37 Durner J, Wendehenne D & Klessig DF (1998) Defense gene induction in tobacco by nitric oxide, cyclic GMP, and cyclic ADP-ribose. Proc Natl Acad Sci USA 95, 10328–10333. 38 Barroso JB, Corpas FJ, Carreras A, Sandalio LM, Valderrama R, Palma JM, Lupianez JA & del Rio LA (1999) Localization of nitric-oxide synthase in plant peroxisomes. J Biol Chem 274, 36729–36733. 39 Delledonne M, Zeier J, Marocco A & Lamb C (2001) Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response. Proc Natl Acad Sci USA 98, 13454– 13459. 40 Lum HK, Butt YK & Lo SC (2002) Hydrogen peroxide induces a rapid production of nitric oxide in mung bean (Phaseolus aureus). Nitric Oxide 6, 205–213. 41 Neill SJ, Desikan R, Clarke A & Hancock JT (2002) Nitric oxide is a novel component of abscisic acid sig- naling in stomatal guard cells. Plant Physiol 128, 13–16. 42 Guo FQ, Okamoto M & Crawford NM (2003) Identifi- cation of a plant nitric oxide synthase gene involved in hormonal signaling. Science 302, 100–103. 43 Melotto M, Underwood W, Koczan J, Nomura K & 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. 51 Kim SC & Raushel FM (1986) Isotopic probes of the argininosuccinate lyase reaction. Biochemistry 25, 4744– 4749. 52 Rosenthal GA & Naylor AW (1969) Purification and general properties of argininosuccinate lyase from jack bean, Canavalia ensiformis (L.) DC. Biochem J 112, 415–419. 53 O’Brien WE & Barr RH (1981) Argininosuccinate lyase: purification and characterization from human liver. Biochemistry 20, 2056–2060. 54 Chia DW, Yoder TJ, Reiter WD & Gibson SI (2000) Fumaric acid: an overlooked form of fixed carbon in Arabidopsis and other plant species. Planta 211, 743– 751. 55 Hurth MA, Suh SJ, Kretzschmar T, Geis T, Bregante M, Gambale F, Martinoia E & Neuhaus HE (2005) Impaired pH homeostasis in Arabidopsis lacking the vacuolar dicarboxylate transporter and analysis of carboxylic acid transport across the tonoplast. Plant Physiol 137, 901–910. 56 Knowles RG & Salter M (1998) Measurement of NOS activity by conversion of radiolabeled arginine to citrul- line using ion-exchange separation. Methods Mol Biol 100, 67–73. R. Tischner et al. Argininosuccinate lyase and NOS assay FEBS Journal 274 (2007) 4238–4245 ª 2007 The Authors Journal compilation ª 2007 FEBS 4245 . Interference with the citrulline-based nitric oxide synthase assay by argininosuccinate lyase activity in Arabidopsis extracts Rudolf Tischner 1, *,. FEBS Argininosuccinate is the immediate precursor to arginine in the urea cycle, and is converted to arginine and fumarate by argininosuccinate lyase (ASL; EC