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• W2143399007 abstract "In mammalian cells, induced expression of arginase in response to wound trauma and pathogen infection plays an important role in regulating the metabolism of l-arginine to either polyamines or nitric oxide (NO). In higher plants, which also utilize arginine for the production of polyamines and NO, the potential role of arginase as a control point for arginine homeostasis has not been investigated. Here, we report the characterization of two genes (LeARG1 and LeARG2) from Lycopersicon esculentum (tomato) that encode arginase. Phylogenic analysis showed that LeARG1 and -2, like all other plant arginases, are more similar to agmatinase than to arginases from vertebrates, fungi, and bacteria. Never-theless, recombinant LeARG1 and -2 exhibited specificity for l-arginine over agmatine and related guanidino substrates. The plant enzymes, like mammalian arginases, were inhibited (Ki ∼ 14 μm) by the NO precursor NG-hydroxy-l-arginine. These results indicate that plant arginases define a distinct group of ureohydrolases that function as authentic l-arginases. LeARG1 and LeARG2 transcripts accumulated to their highest levels in reproductive tissues. In leaves, LeARG2 expression and arginase activity were induced in response to wounding and treatment with jasmonic acid (JA), a potent signal for plant defense responses. Wound- and JA-induced expression of LeARG2 was not observed in the tomato jasmonic acid-insensitive1 mutant, indicating that this response is strictly dependent on an intact JA signal transduction pathway. Infection of wild-type plants with a virulent strain of Pseudomonas syringae pv. tomato also up-regulated LeARG2 expression and arginase activity. This response was mediated by the bacterial phytotoxin coronatine, which exerts its virulence effects by co-opting the host JA signaling pathway. These results highlight striking similarities in the regulation of arginase in plants and animals and suggest that stress-induced arginase may perform similar roles in diverse biological systems. In mammalian cells, induced expression of arginase in response to wound trauma and pathogen infection plays an important role in regulating the metabolism of l-arginine to either polyamines or nitric oxide (NO). In higher plants, which also utilize arginine for the production of polyamines and NO, the potential role of arginase as a control point for arginine homeostasis has not been investigated. Here, we report the characterization of two genes (LeARG1 and LeARG2) from Lycopersicon esculentum (tomato) that encode arginase. Phylogenic analysis showed that LeARG1 and -2, like all other plant arginases, are more similar to agmatinase than to arginases from vertebrates, fungi, and bacteria. Never-theless, recombinant LeARG1 and -2 exhibited specificity for l-arginine over agmatine and related guanidino substrates. The plant enzymes, like mammalian arginases, were inhibited (Ki ∼ 14 μm) by the NO precursor NG-hydroxy-l-arginine. These results indicate that plant arginases define a distinct group of ureohydrolases that function as authentic l-arginases. LeARG1 and LeARG2 transcripts accumulated to their highest levels in reproductive tissues. In leaves, LeARG2 expression and arginase activity were induced in response to wounding and treatment with jasmonic acid (JA), a potent signal for plant defense responses. Wound- and JA-induced expression of LeARG2 was not observed in the tomato jasmonic acid-insensitive1 mutant, indicating that this response is strictly dependent on an intact JA signal transduction pathway. Infection of wild-type plants with a virulent strain of Pseudomonas syringae pv. tomato also up-regulated LeARG2 expression and arginase activity. This response was mediated by the bacterial phytotoxin coronatine, which exerts its virulence effects by co-opting the host JA signaling pathway. These results highlight striking similarities in the regulation of arginase in plants and animals and suggest that stress-induced arginase may perform similar roles in diverse biological systems. l-Arginine is one of the most functionally diverse amino acids in living cells. In addition to serving as a constituent of proteins, arginine is a precursor for the biosynthesis of polyamines, agmatine, and proline, as well as the cell-signaling molecules glutamate, γ-aminobutyric acid, and nitric oxide (1Wu G.Y. Morris S.M. Biochem. J. 1998; 336: 1-17Crossref PubMed Scopus (2157) Google Scholar, 2Morris S.M. Annu. Rev. Nutr. 2002; 22: 87-105Crossref PubMed Scopus (481) Google Scholar, 3Cederbaum S.D. Yu H. Grody W.W. Kern R.M. Yoo P. Iyer R.K. Mol. Genet. Metab. 2004; 81: S38-S44Crossref PubMed Scopus (209) Google Scholar). Two of the most intensively studied pathways of arginine metabolism are those catalyzed by arginase and nitric-oxide synthase (NOS). 1The abbreviations used are: NOS, nitric-oxide synthase; l-NOHA, NG-hydroxy-l-arginine; LeARG, Lycopersicon esculentum arginase; JA, jasmonic acid; MeJA, methyl jasmonate; Pst, Pseudomonas syringae pv. tomato; COR, coronatine; EST, expressed sequence tag; ODC, ornithine decarboxylase; ADC, arginine decarboxylase; COI1, coronatine-insensitive 1; CHES, 2-(cyclohexylamino)ethanesulfonic acid; TIGR, The Institute for Genomic Research; dpi, days post-infection.1The abbreviations used are: NOS, nitric-oxide synthase; l-NOHA, NG-hydroxy-l-arginine; LeARG, Lycopersicon esculentum arginase; JA, jasmonic acid; MeJA, methyl jasmonate; Pst, Pseudomonas syringae pv. tomato; COR, coronatine; EST, expressed sequence tag; ODC, ornithine decarboxylase; ADC, arginine decarboxylase; COI1, coronatine-insensitive 1; CHES, 2-(cyclohexylamino)ethanesulfonic acid; TIGR, The Institute for Genomic Research; dpi, days post-infection. Arginase hydrolyzes arginine to urea and ornithine, the latter of which is a precursor for polyamine biosynthesis. Recent studies in animal systems indicate that increased arginase expression stimulates the production of polyamines that promote tumor cell proliferation (4Chang C.I. Liao J.C. Kuo L. Cancer Res. 2001; 61: 1100-1106PubMed Google Scholar), wound healing (5Satriano J. Ann. N. Y. Acad. Sci. 2003; 1009: 34-43Crossref PubMed Scopus (67) Google Scholar), and axonal regeneration following injury (6Cai D. Deng K. Mellado W. Lee J. Ratan R.R. Filbin M.T. Neuron. 2002; 35: 711-719Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Jux-taposed to the growth-promoting effects of polyamines are the cytostatic effects of NO produced by activated macrophages. The switch between the arginase and NOS branches of arginine metabolism is controlled by various inflammatory signals that regulate arginase expression and arginine availability (2Morris S.M. Annu. Rev. Nutr. 2002; 22: 87-105Crossref PubMed Scopus (481) Google Scholar, 7Lee J. Ryu H. Ferrante R.J. Morris Jr., S.M. Ratan R.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4843-4848Crossref PubMed Scopus (285) Google Scholar, 8Bronte V. Serafini P. Mazzoni A. Segal D.M. Zanovello P. Trends Immunol. 2003; 24: 302-306Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, 9Hallemeesch M.M. Lamers W.H. Deutz N.E. Clin. Nutr. 2002; 21: 273-279Abstract Full Text PDF PubMed Scopus (66) Google Scholar). Because arginase and NOS compete for a common substrate, increased arginase expression can effectively attenuate the NOS pathway, often with profound physiological consequences. A diversity of human pathogens, for example, induce arginase expression as a means of evading NO-mediated host defenses (10Duleu S. Vincendeau P. Courtois P. Semballa S. Lagroye I. Daulouede S. Boucher J.L. Wilson K.T. Veyret B. Gobert A.P. J. Immunol. 2004; 172: 6298-6303Crossref PubMed Scopus (67) Google Scholar, 11Gobert A.P. McGee D.J. Akhtar M. Mendz G.L. Newton J.C. Cheng Y. Mobley H.L. Wilson K.T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13844-13849Crossref PubMed Scopus (310) Google Scholar, 12Iniesta V. Gomez-Nieto L.C. Molano I. Mohedano A. Carcelen J. Miron C. Alonso C. Corraliza I. Parasite Immunol. 2002; 24: 113-118Crossref PubMed Scopus (130) Google Scholar, 13Vincendeau P. Gobert A.P. Daulouede S. Moynet D. Mossalayi M.D. Trends Parasitol. 2003; 19: 9-12Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The interaction between the arginase and NOS pathways extends beyond the fact that they both use a common substrate. For example, the intermediate in the NOS-catalyzed production of NO, NG-hydroxy-l-arginine (NOHA), functions as a potent inhibitor of arginase (14Boucher J.L. Custot J. Vadon S. Delaforge M. Lepoivre M. Tenu J.P. Yapo A. Mansuy D. Biochem. Biophys. Res. Commun. 1994; 203: 1614-1621Crossref PubMed Scopus (149) Google Scholar, 15Daghigh F. Fukuto J.M. Ash D.E. Biochem. Biophys. Res. Commun. 1994; 202: 174-180Crossref PubMed Scopus (161) Google Scholar). In contrast to our understanding of arginase regulation in animals, very little is known about the potential role of arginase as a metabolic control point for arginine homeostasis in higher plants. The well established role of NO in plant developmental and defense-related processes (16Durner J. Klessig D.F. Curr. Opin. Plant Biol. 1999; 2: 369-374Crossref PubMed Scopus (383) Google Scholar, 17Wendehenne D. Pugin A. Klessig D.F. Durner J. Trends Plant Sci. 2001; 6: 177-183Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, 18McDowell J.M. Dangl J.L. Trends Biochem. Sci. 2000; 25: 79-82Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar), together with the recent discovery of two arginine-utilizing plant NOSs (19Chandok M.R. Ytterberg A.J. van Wijk K.J. Klessig D.F. Cell. 2003; 113: 469-482Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 20Guo F.Q. Okamoto M. Crawford N.M. Science. 2003; 302: 100-103Crossref PubMed Scopus (709) Google Scholar), provides a strong rationale for addressing this question. Most studies of plant arginase have focused on its role in mobilizing arginine as a nitrogen source during post-germinative growth (21Splittstoesser W.E. Phytochemistry. 1969; 8: 753-758Crossref Scopus (32) Google Scholar, 22Kollöffel C. van Dijke H.D. Plant Physiol. 1975; 55: 507-510Crossref PubMed Google Scholar, 23Wright L.C. Brady C.J. Hinde R.W. Phytochemistry. 1981; 20: 2641-2645Crossref Scopus (22) Google Scholar, 24Boutin J.P. Eur. J. Biochem. 1982; 127: 237-243Crossref PubMed Scopus (26) Google Scholar, 25Kang J.H. Cho Y.D. Plant Physiol. 1990; 93: 1230-1234Crossref PubMed Scopus (42) Google Scholar, 26Polacco J.C. Holland M.A. Int. Rev. Cytol. 1993; 145: 65-103Crossref Scopus (118) Google Scholar, 27Carvajal N. Olave N. Salas M. Uribe E. Enríquez S. Phytochemistry. 1996; 41: 373-376Crossref Scopus (10) Google Scholar, 28Hwang H.J. Kim E.H. Cho Y.D. Phytochemistry. 2001; 58: 1015-1024Crossref PubMed Scopus (29) Google Scholar). Arginine can account for 50% of the nitrogen in seed protein, and up to 90% of the free nitrogen in vegetative tissues. In several plant species, including soybean, broad bean, pumpkin, Arabidopsis, and loblolly pine, nitrogen mobilization during seedling development is correlated with large increases in arginase expression (26Polacco J.C. Holland M.A. Int. Rev. Cytol. 1993; 145: 65-103Crossref Scopus (118) Google Scholar, 28Hwang H.J. Kim E.H. Cho Y.D. Phytochemistry. 2001; 58: 1015-1024Crossref PubMed Scopus (29) Google Scholar, 29Todd C.D. Gifford D.J. Planta. 2002; 215: 110-118Crossref PubMed Scopus (21) Google Scholar). Seedling arginase catalyzes the breakdown of a significant portion of the arginine pool to ornithine and urea. Ornithine can support the biosynthesis of polyamines, proline, and glutamate, whereas urea is further catabolized by urease to carbon dioxide and ammonium. The coordinate action of arginase and urease is thought to recycle urea nitrogen to meet the metabolic demands of developing seedlings (26Polacco J.C. Holland M.A. Int. Rev. Cytol. 1993; 145: 65-103Crossref Scopus (118) Google Scholar, 30Zonia L.E. Stebbins N.E. Polacco J.C. Plant Physiol. 1995; 107: 1097-1103Crossref PubMed Scopus (184) Google Scholar). The molecular mechanisms by which arginase expression in plants is regulated by developmental or stress-related cues remain to be determined. A prerequisite for addressing this question is the unambiguous identification of genes that encode plant arginase. cDNAs encoding putative arginases has been reported for Arabidopsis (31Krumpelman P.M. Freyermuth S.K. Cannon J.F. Fink G.R. Polacco J.C. Plant Physiol. 1995; 107: 1479-1480Crossref PubMed Scopus (29) Google Scholar), soybean (32Goldraij A. Polacco J.C. Plant Physiol. 1999; 119: 297-304Crossref PubMed Scopus (60) Google Scholar), and loblolly pine (33Todd C.D. Cooke J.E. Mullen R.T. Gifford D.J. Plant Mol. Biol. 2001; 45: 555-565Crossref PubMed Scopus (38) Google Scholar). The arginase superfamily is composed of enzymes that hydrolyze various guanidino substrates to a one-carbon nitrogen-containing product (e.g. urea) and a second product that retains the quaternary nitrogen at the site of hydrolysis. The family includes arginase, agmatinase, proclavaminate amidinohydrolase, formiminoglutamase, as well as several uncharacterized sequences from archaea and eubacteria (34Perozich J. Hempel J. Morris Jr., S.M. Biochim. Biophys. Acta. 1998; 1382: 23-37Crossref PubMed Scopus (90) Google Scholar, 35Sekowska A. Danchin A. Risler J.L. Microbiology-UK. 2000; 146: 1815-1828Crossref PubMed Scopus (54) Google Scholar). Because the predicted sequences of plant arginases are more similar to agmatinase and other arginase-like enzymes than to non-plant arginases from vertebrates, fungi, and bacteria, it was suggested that plant genes annotated as arginase may encode agmatinase or another amidinohydrolase activity involved in the production of secondary metabolites (34Perozich J. Hempel J. Morris Jr., S.M. Biochim. Biophys. Acta. 1998; 1382: 23-37Crossref PubMed Scopus (90) Google Scholar, 35Sekowska A. Danchin A. Risler J.L. Microbiology-UK. 2000; 146: 1815-1828Crossref PubMed Scopus (54) Google Scholar). Although an Arabidopsis arginase cDNA can genetically complement an arginase-deficient yeast mutant (31Krumpelman P.M. Freyermuth S.K. Cannon J.F. Fink G.R. Polacco J.C. Plant Physiol. 1995; 107: 1479-1480Crossref PubMed Scopus (29) Google Scholar), no direct enzymatic data have been reported for the product of any plant arginase gene. To begin to assess the role of arginase in arginine homeostasis in higher plants, we identified and characterized two arginase genes (LeARG1 and LeARG2) from tomato. Our results demonstrate that, despite their phylogenetic similarity to agmatinases, the proteins encoded by LeARG1 and LeARG2 have robust amidinohydrolase activity against and high specificity for l-arginine. We report that LeARG2 expression in leaves is strongly induced by wounding and, furthermore, that this induction is mediated by the plant stress signal jasmonic acid (JA). We also document induced expression of arginase in response to Pseudomonas syringae, the causal agent of bacterial speck disease. The bacterial toxin coronatine, which exerts its effects by activating the host JA signaling pathway, was both necessary and sufficient for arginase induction in P. syringae-infected plants. The potential role of stress-induced arginase in higher plants is discussed. Plant Material and Treatments—Tomato (Lycopersicon esculentum cv. Castlemart) plants were grown in Jiffy peat pots (Hummert International) in a growth chamber maintained under 17 h of light (200 μE m-2 s-1) at 28 °C and 7 h of dark at 18 °C. Seed for the sterile jai1-1 mutant was obtained from a segregating population as described by Li et al. (36Li L. Li C. Howe G.A. Plant Physiol. 2001; 127: 1414-1417Crossref PubMed Scopus (93) Google Scholar). Flowers and fruits were harvested from plants maintained in a greenhouse. For experiments involving methyl-JA (MeJA) treatment, 3-week-old plants were placed in a closed Lucite box (31 cm × 27 cm × 14 cm) and treated with 2 μl of pure MeJA (Bedoukian Research) dissolved in 300 μl of ethanol, as previously described (37Li L. Howe G.A. Plant Mol. Biol. 2001; 46: 409-419Crossref PubMed Scopus (40) Google Scholar). A hemostat was used to inflict mechanical wounds near the distal end of leaflet, perpendicular to the midvein. Zhao et al. (38Zhao Y. Thilmony R. Bender C.L. Schaller A. He S.Y. Howe G.A. Plant J. 2003; 36: 485-499Crossref PubMed Scopus (265) Google Scholar) described the source of coronatine (COR) and application to tomato plants. Briefly, 20 ng of COR (dissolved in 0.1 m NH4HCO3, 5 ng/μl) was applied to the adaxial surface of leaflets of 3-week-old tomato plants. Control plants were treated with 4 μlof0.1 m NH4HCO3. The sources and growth conditions of Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) and the mutant strain Pst DC3118 COR- were described previously (38Zhao Y. Thilmony R. Bender C.L. Schaller A. He S.Y. Howe G.A. Plant J. 2003; 36: 485-499Crossref PubMed Scopus (265) Google Scholar). Bacterial suspensions were vacuum-infiltrated into the leaves of 3-week-old plants (38Zhao Y. Thilmony R. Bender C.L. Schaller A. He S.Y. Howe G.A. Plant J. 2003; 36: 485-499Crossref PubMed Scopus (265) Google Scholar). Three replicate samples were taken for each treatment over a 4-day period. At various times following the treatment, leaf tissue was harvested, frozen in liquid nitrogen, and stored at -80 °C until further use for RNA extraction or arginase assays (see below). Identification of Full-length LeARG cDNAs—A search of the tomato EST (Expressed Sequence Tag) data base (version 9.0 released on April 17, 2003) at the Institute for Genomic Research (www.tigr.org/tdb/lgi/) identified two tentative consensus sequences (TC124738 and TC124737) that were annotated as arginase. cDNA clones (EST435583 and EST337938) corresponding to representative members of these two genes were obtained from the Clemson University Genomics Institute. cDNA inserts from each clone were sequenced in their entirety on both strands. The cDNA corresponding to EST435583, which we designated LeARG1, was 1508 bp in length and included 252 bp upstream of the initiator AUG codon and 209 bp in the 3′-untranslated region (excluding 30 poly(A) residues). The presence of an in-frame stop codon (TAA) nine nucleotides upstream of the initiator AUG codon indicated that the cDNA encodes a full-length protein. The cDNA corresponding to EST337938, which we designated LeARG2, was 1360 bp in length and included 19 bp upstream of the initiator AUG codon and 266 bp in the 3′-untranslated region (excluding 58 poly(A) residues). The presence of an in-frame stop codon (TAA) 9 nucleotides upstream of the initiator AUG codon indicated that this cDNA also encodes a full-length protein. Data base searches were performed using the BLAST program (39Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69707) Google Scholar) available at the U.S. National Center for Biotechnology. Arginase Phylogeny—Members of the arginase superfamily were identified by BLAST searches against non-redundant sequence databases (www.ncbi.nlm.nih.gov/BLAST/) and TIGR plant EST databases (www.tigr.org/tdb/tgi/plant.shtml). Sequences obtained from the TIGR databases are composed of unigene clusters of multiple EST clones. A total of 85 sequences were used for construction of the phylogenetic tree (Fig. 1). Sequence accession numbers are listed in Fig. S1 (Supplemental Materials). Amino acid sequences were aligned using PILEUP in the GCG software suite (Wisconsin Package version 10.2, Genetics Computer Group (GCG), Madison, WI). A neighbor-joining phylogeny was constructed from mean character distances using PAUP 4.0*, version 4.0b10 (40Swofford D.L. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods), Version 4. Sinauer Associates, Sunderland, MA2000Google Scholar). Neighbor-joining bootstrap replicates were run to test the branching order reliability. Expression and Purification of Recombinant LeARG1 and LeARG2— Basic molecular techniques were performed as described in Sambrook et al. (41Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Springer Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). A PCR-based approach was used to construct the expression vector that added a C-terminal His6 tag to the LeARG coding sequence. Forward and reverse primers were designed to contain NdeI and XhoI restriction sites, respectively. For preparation of the LeARG1 construct, the sequence of the forward primer was 5′-GGA ATT CCA TAT GAG GAG TGC TGG AAG AAT-3′ and that of the reverse primer was 5′-CCG CTC GAG CTT GGA TAT CTT GGC AGT AAG-3′. For preparation of the LeARG2 construct, the sequence of the forward primer was 5′-GGA ATT CCA TAT GAA GAG TGC TGG AAG TAT-3′ and that of the reverse primer was 5′-CCG CTC GAG CTT GGA CAT CTT GGC AGC AAG-3′. PCR amplification of EST435583 (LeARG1) and EST337938 (LeARG2) yielded a 1.0-kilobase product that was subsequently cut with NdeI and XhoI subcloned into the same sites of the expression vector pET-23b (Novagen, Madison, WI). The resulting construct placed an additional eight amino acids (LEHHHHHH) on the C terminus of the protein. His-tagged recombinant proteins were expressed in BL21(DE3) host cells as follows. An overnight culture (1 ml) was inoculated into 50 ml of Terrific Broth medium supplemented with 200 μg/ml ampicillin. Bacteria were grown at 37 °C in a shaker at 250 rpm for 4 h to a cell density of about 1.2 A600, and then isopropyl 1-thio-β-d-galactopyranoside was added to a final concentration of 0.25 mm. The induced culture was incubated for 4 h at 37 °C. Cells were collected by centrifugation and stored at -20 °C until further use. Purification of the His-tagged LeARG1 and LeARG2 was performed at 4 °C except where otherwise noted. Bacterial cells expressing the construct were harvested from 50 ml of culture medium, followed by resuspension in 2 ml of Tris buffer (50 mm, pH 8.0) containing 0.1 mm phenylmethylsulfonyl fluoride. Cells were first incubated with 2.5 mg of lysozyme for 60 min at room temperature and then lysed using three 2-min pulses from a probe-type sonicator (Branson Sonifier Model 450). Cell homogenates were centrifuged at 20,000 × g for 10 min. The resulting supernatant was collected, and the buffer was exchanged to binding buffer (5 mm imidazole, 500 mm NaCl, 20 mm Tris-HCl, pH 7.9) with a 5-ml spin column prepared with Sephadex G-25 (Amersham Biosciences) and equilibrated with binding buffer. Nickel-charged resin columns having a 1-ml bed volume (Qiagen) were conditioned with 10 ml of water and then 5 ml of binding buffer. After loading the protein solution (2 ml in binding buffer), the column was washed with 10 ml of binding buffer and 10 ml of washing buffer (80 mm imidazole, 500 mm NaCl, 20 mm Tris-HCl, pH 7.9). His-tagged arginase was eluted with elution buffer (400 mm imidazole, 500 mm NaCl, 20 mm Tris-HCl, pH 7.9) and collected in 2-ml fractions. Arginase eluted in the first two fractions as determined by analysis of fractions on SDS-polyacrylamide gels. Imidazole was removed from the protein samples with a 5-ml spin column packed with Sephadex G-25 and equilibrated with 100 mm Tris-HCl buffer (pH 7.5). Protein concentrations were determined by the Bradford method (42Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214455) Google Scholar), using bovine serum albumin as a standard. The relative purity of recombinant protein was assessed by SDS-polyacrylamide gel electrophoresis and staining of gels with Coomassie Brilliant Blue R-250. Enzyme Assays—Frozen tomato leaves (∼1.5 g) were ground in liquid nitrogen with a mortar and pestle and then homogenized in 10 ml of 100 mm Tris-HCl (pH 7.5) containing 1% (v/v) 2-mercaptoethanol and 0.1 mm phenylmethylsulfonyl fluoride. Homogenates were centrifuged at 20,000 × g for 10 min at 4 °C, and the supernatants were used as the enzyme source. Recombinant LeARG enzyme was prepared as described above. Protein concentrations were determined as described above. Arginase activity was measured with a spectrophotometric assay for detection of urea (43Alabadí D. Aguero M.S. Perez-Amador M.A. Carbonell J. Plant Physiol. 1996; 112: 1237-1244Crossref PubMed Scopus (67) Google Scholar), with minor modifications. The enzyme solution was activated with 1 mm MnCl2 at 37 °C for 60 min. The reaction mixture (0.5 ml) contained 10 μl of the enzyme source in assay buffer (50 mm CHES buffer (pH 9.6), 250 mm l-arginine, 2 mm MnCl2). Reactions were carried out at 37 °C for 20 min and stopped by the addition of 500 μl of 15% (v/v) perchloric acid. A 200-μl aliquot was mixed vigorously with 3 ml of acid mixture (9% (v/v) of phosphoric acid and 27% (v/v) of sulfuric acid) and 100 μl of 3% (w/v) α-isonitrosopropiophenone (Sigma) in 95% ethanol. This mixture was heated in a boiling water bath in the dark for 60 min and cooled for 10 min to room temperature. The A540 was recorded on a Uvikon 933 spectrophotometer (Research Instruments, San Diego, CA). Substrate specificity tests were performed as described above with the exception that agmatine and other related compounds were added in place of l-arginine, to a final concentration of 250 mm. All substrates tested were obtained from Sigma. Three buffer systems were used to test the effect of pH on arginase activity: 200 mm potassium phosphate, pH 7.0, 7.5, 11.0, and 12.0; 200 mm Tris-HCl, pH 7.5, 8.0, and 8.5; and 200 mm Gly-NaOH, pH 8.7, 9.0, 9.5, 10.0, and 10.5 (Alabadí et al. (43Alabadí D. Aguero M.S. Perez-Amador M.A. Carbonell J. Plant Physiol. 1996; 112: 1237-1244Crossref PubMed Scopus (67) Google Scholar)). Inhibitor studies were conducted with test compounds that were dissolved in water and then diluted into the assay buffer at various concentrations prior to addition of enzyme. For example, 1 μl of a 5-mm l-NOHA stock was added to 489 μl of assay buffer, followed by addition of 10 μl of enzyme solution. The reaction was carried out as described above. l-NOHA was obtained from Cayman Chemical (Ann Arbor, MI). Nucleic Acid Blot Analysis—RNA blot analyses were performed as previously described (44Howe G.A. Lee G.I. Itoh A. Li L. DeRocher A. Plant Physiol. 2000; 123: 711-724Crossref PubMed Scopus (214) Google Scholar). Full-length LeARG1 and LeARG2 cDNAs were PCR-amplified with T3 and T7 primers that anneal to the pBlueScript vector. Because full-length LeARG1 and LeARG2 cDNAs cross-hybridize to each other, a PCR-based approach was used to generate gene-specific probes corresponding to the diverged untranslated regions of the cDNA. Primers used to generate the LeARG1-specific probe were 5′-CCC CTT CAC AAG AGA AGA AAT-3′ and 5′-TTC TGA TTA TCC TAC AAC TGC-3′. The resulting 233-bp product hybridizes to the 5′-untranslated region of LeARG1 transcripts. Primers used to generate the LeARG2-specific probe were 5′-CAA GCA AGA AGT ACC ATG TAT-3′ and T7 5′-TAA TAC GAC TCA CTA TAG GG-3′ (T7 primer), which gave a 349-bp product that included 48 bp from the pBluescript SK vector. This probe hybridized specifically to the 3′-untranslated region of LeARG2 transcripts. Total RNA was extracted from various tissues of soil-grown plants. Hybridization signals on RNA blots were normalized to the signal obtained using a cDNA probe for translation initiation factor eIF4A mRNA, obtained from Clemson University (EST clone cLED1D24). Tomato genomic DNA preparations and Southern blot analysis were as described previously (44Howe G.A. Lee G.I. Itoh A. Li L. DeRocher A. Plant Physiol. 2000; 123: 711-724Crossref PubMed Scopus (214) Google Scholar). Identification of Arginase-encoding Genes in Tomato—A search of the tomato EST data base at The Institute for Genomic Research (TIGR) identified two genes annotated as arginase, which we designated LeARG1 and LeARG2. Full-length LeARG1 and LeARG2 cDNAs were predicted to encode 338-amino acid proteins having calculated molecular weights of 37,048 and 36,851, respectively. The deduced amino acid sequences of LeARG1 and LeARG2 were 89% identical to each other, and 70–87% identical to arginase sequences reported from Arabidopsis (31Krumpelman P.M. Freyermuth S.K. Cannon J.F. Fink G.R. Polacco J.C. Plant Physiol. 1995; 107: 1479-1480Crossref PubMed Scopus (29) Google Scholar), soybean (32Goldraij A. Polacco J.C. Plant Physiol. 1999; 119: 297-304Crossref PubMed Scopus (60) Google Scholar), and loblolly pine (33Todd C.D. Cooke J.E. Mullen R.T. Gifford D.J. Plant Mol. Biol. 2001; 45: 555-565Crossref PubMed Scopus (38) Google Scholar). Sequence alignments between the plant arginases showed a high degree of amino acid sequence identity over almost the entire length of the polypeptide. The greatest sequence diversity between plant arginases was at the N-terminal end. This region of the plant arginase exhibits features of a mitochondrial targeting peptide (32Goldraij A. Polacco J.C. Plant Physiol. 1999; 119: 297-304Crossref PubMed Scopus (60) Google Scholar), which is consistent with the localization of the enzyme to this organelle (22Kollöffel C. van Dijke H.D. Plant Physiol. 1975; 55: 507-510Crossref PubMed Google Scholar, 32Goldraij A. Polacco J.C. Plant Physiol. 1999; 119: 297-304Crossref PubMed Scopus (60) Google Scholar). Amino acid sequence alignments between 85 amidinohydrolases from diverse organisms indicated that the arginase superfamily is divided into four major groups: (i) l-arginases from vertebrates, fungi, and bacteria (referred to here as non-plant arginases); (ii) plant arginases, including LeARG1 and LeARG2; (iii) agmatinases and agmatinase-like enzymes; and (iv) several hypothetical arginase-like proteins from archea and eubacteria (Fig. 1). All plant arginases (14 sequences from 11 species) formed a monophyletic cluster that was clearly distinguisha" @default.
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• W2143399007 date "2004-10-01" @default.
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• W2143399007 title "Regulation of Plant Arginase by Wounding, Jasmonate, and the Phytotoxin Coronatine" @default.
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• W2143399007 doi "https://doi.org/10.1074/jbc.m407151200" @default.
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