Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Journal of Experimental Botany, Vol. 50, No. 330, pp. 63–69, January 1999 Aldehyde oxidase in roots, leaves and seeds of barley (Hordeum vulgare L.) Rustem T. Omarov1,3, Shuichi Akaba2, Tomokazu Koshiba2 and S. Herman Lips1 1 Biostress Research Laboratory (J. Blaustein Institute for Desert Researches) and Department of Life Sciences (Faculty of Natural Sciences), Ben-Gurion University of the Negev, Sede Boqer 84990, Israel 2 Department of Biology, Tokyo Metropolitan University, Hachioji-shi, Tokyo 192–0397, Japan Received 8 April 1998; Accepted 19 August 1998 Abstract Aldehyde oxidase (AO, EC 1.2.3.1) proteins in leaves, roots and seeds of barley (Hordeum vulgare L.) plants were studied. Differences in substrate specificity and mobility in native PAGE between AO proteins extracted from roots, leaves and seeds have been observed. Four clear bands of AO reacting proteins were detected in barley plants capable of oxidizing a number of aliphatic and aromatic aldehydes such as indole-3-aldehyde, acetaldehyde, heptaldehyde, and benzaldehyde. Mouse polyclonal antibodies raised against purified maize AO cross-reacted with barley AO proteins extracted from roots, leaves and seeds. At least three different AO proteins were detected in roots on the basis of their mobility during PAGE after native Western blot analysis while in leaves and seeds only one polypeptide cross-reacted with the antibody. SDS-immunoblot analysis showed marked differences in molecular weight between subunits of the AO bands extracted from roots, leaves and seeds. Two distinct subunit bands were observed in roots, with relatively close molecular weights (160 kDa and 145 kDa), while a single subunit with a molecular weight of 150 kDa was observed in leaf and seed extracts. Menadione, a specific and potent inhibitor of animal AO did not affect barley AO proteins. Root and leaf AO differed in their thermostability and susceptibility to exogenous tungstate. The AO proteins in plants may be a group of enzymes with different substrate specificity, tissue distribution and presumably fulfilling different metabolic roles in each plant organ. Key words: Aldehyde oxidase, barley, roots, leaves, seeds, molybdenum cofactor (MoCo). Introduction AO (EC 1.2.3.1) is a member of the family of molybdenum hydroxylases, iron sulphur flavoproteins involved in the metabolism of a broad range of natural and xenobiotic compounds, most of which have been studied in animals and microorganisms. Early observations demonstrated the presence of AO activities in oat coleoptiles (Rajagopal, 1971), potato tubers (Rothe, 1974), cucumber seedlings (Bower et al., 1978), and pea seedlings (Miyata et al., 1981). Much of the interest in AO in plants stems from its involvement in the biosynthesis of abscisic acid (ABA) and indole acetic acid (IAA). The enzyme seems to catalyse the last step of the biosynthesis of these hormones, through the oxidation of indole3-acetaldehyde (IAAld ) to IAA ( Koshiba et al., 1996) and abscisic aldehyde (ABAld) to ABA ( Taylor et al., 1988). Molybdenum cofactor (MoCo)-deficient mutants of barley and tobacco lacked AO and XDH activities and were almost totally impaired in their capacity to produce ABA ( Walker-Simmons et al., 1989; Leydecker et al., 1995). The flacca mutant of tomato lacks aldehyde oxidase and xanthine dehydrogenase, but not nitrate reductase (Marin and Marion-Poll, 1997) suggesting that the expression of the genes responsible for the synthesis of MoCo is unaffected. The aba3 mutant of Arabidopsis (Arabidopsis thaliana) may be affected in its capacity to introduce a third sulphur atom into the Mo-centre of AO and XDH (Schwartz et al., 1997). Similarly, the mutants 3 To whom correspondence should be addressed. Fax: +972 7 6596752. E-mail: [email protected] © Oxford University Press 1999 64 Omarov et al. aba1 in tobacco and flacca in tomato may not be affected in their capacity to produce MoCo but rather on the sulphurylation of the Mo hydroxylases. Maize (Zea mays L.) coleoptile AO has been reported to be a flavin- and molybdenum-containing enzyme with a molecular mass of 300 kDa composed of two identical subunits of 150 kDa ( Koshiba et al., 1996). A leaf AO of citrus (Citrus sinensis L.) catalysed the oxidation of indole-3-aldehyde to indole-3-carboxylic acid ( Winer et al., 1993). Maize AO has been found to have biochemical properties similar to the animal enzyme ( Koshiba et al., 1996). The tomato AO protein exhibited a considerable amount of amino acid sequence homology to AO and xanthine dehydrogenase of various organisms (Ori et al., 1997). Two cDNAs for maize AO (zmAO-1 and zmAO-2) have been cloned from maize coleoptiles (Sekimoto et al., 1997). On the basis of their very high homology it was suggested that both AO proteins were related but may play different roles in the plant. Recently four cDNAs (atAO-1, 2, 3 and 4) encoding MoCo-containing AOs have been cloned in Arabidopsis (Sekimoto et al., 1998). Limited information is available on AO distribution in different plant organs and little is known about its physiological roles. It has been reported recently that activity staining of Arabidopsis seedlings AO after native PAGE revealed localization of AO in an organ-specific manner (Seo et al., 1998). In Arabidopsis at AO-1 and at AO-2 genes were expressed at higher levels in lower hypocotyls and roots of seedlings, while at AO-3 was slightly higher in cotyledons and upper hypocotyls (Sekimoto et al., 1998). Root AO but not the leaf enzyme was enhanced by salinity in barley plants, especially in the presence of ammonium in the nutrient medium (Omarov et al., 1998). AO activity increased remarkably in roots and shoots of ryegrass plants following applications of salinity and ammonium to nutrient medium (Sagi et al., 1998). In the present paper, some biochemical properties and differences of AO proteins in roots and leaves of barley are described using native PAGE, Western blot analysis and other assays. The main goal of the present study is the characterization of different AO proteins observed in plant organs and, eventually, to define their principal specific functions in each part of the plant. Materials and methods Plant material Barley (Hordeum vulgare L.) cv. Steptoe seedlings were grown in 20 l containers with nutrient solutions as previously described (Savidov et al., 1997a). The seeds were germinated in 0.2 mM CaSO in the dark at room temperature over 3 d. Uniform 4 plants, 23 pot−1, were grown in 4–8 replications for each treatment in a completely randomized block design. The experiments were conducted in a greenhouse, with average day temperatures between 20 °C and 25 °C during the day, and between 8 °C and 12 °C during the night. Midday PPFD was 900–1000 mmol m−2 s−1. NH Cl at 4 mM was supplied as the 4 nitrogen source in half-strength modified Hoagland nutrient solution (Hoagland and Arnon, 1938) in order to enhance AO activity in roots (Omarov et al, 1998). Plants grown in vermiculite were irrigated twice daily with identical volumes of nutrient solution to prevent an increase of more than 10% in the EC of the leachate as compared with that of the input solution. Extraction and assay of AO Barley roots, leaves and seeds were frozen in liquid nitrogen immediately after harvesting and ground in an ice-cold mortar with acid-washed sand and ice-cold extraction medium. The extraction medium contained 250 mM TRIS-HCl (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, 5 mM reduced glutathione (GSH ), 5 mM FAD, and 50 mM leupeptin. The tissue/buffer ratios were 152 (w/v) for roots and leaves and 156 (w/v) for seed material. The homogenized plant material was centrifuged in a Centrikon T-124 refrigerated centrifuge at 27 000 g at 4 °C for 15 min. The resulting supernatant was used in subsequent assays. AO activity was detected in polyacrylamide gels by staining after native electrophoresis. Native gels were prepared with 7.5% acrylamide gel (Laemmli, 1970) in the absence of SDS at 4 °C. The gel was immersed after electrophoresis in 0.2 M phosphate buffer, pH 7.5, for 10 min followed by gentle shaking at room temperature in a reaction mixture containing 0.1 M TRIS-HCl, pH 8.0, 0.1 mM phenazine methosulphate, 1 mM MTT (3[4,5-dimethylthiazol-2-yl ]-2,5-diphenyltetrazoliumbromide) and 1 mM substrate (heptaldehyde, indole-3-aldehyde, benzaldehyde or acetaldehyde). Native PAGE was carried out with a Protean II xi Cell (Bio-Rad, USA). Western blot analysis and protein determination The AO proteins extracted from the plant material were subjected to Western blotting. Ground tissue (1 g FW ) was extracted with 3 ml TRIS-HCl buffer, pH 7.5, 3 mM DTT, 1 mM EDTA, 5 mM GSH, and 50 mM leupeptin. After centrifugation of part of the crude extract, the resulting supernatant was added to SDS-buffer at a ratio of 154 (v/v). Native-PAGE was carried out as described above. SDSPAGE was performed in a 10% polyacrylamide gel (Laemmli, 1970). The resulting gel with the separated proteins was then electrophoretically transferred on to a nitrocellulose membrane (0.2 mm pore size; Schleicher and Schüll, Dassel, Germany). Blotting time was 1 h at 2 mA cm−2. Blots were blocked for 90 min in 5% (w/v) bovine serum albumin in TBS. Immunodetection of AO was carried out with polyclonal mouse antibodies raised against the purified maize AO ( Koshiba et al., 1996) after a 500-fold dilution in TBS and secondary antibodies (anti-mouse IgG, Sigma) diluted 1000-fold in TBS. The antigen/ primary antibody complex was detected by binding of alkalinephosphatase-linked goat anti-mouse IgG (Sigma, USA). Phosphatase activity was developed by staining with 5-bromo4-chloro-3-indolyl phosphate and nitroblue tetrazolium (BCIP/NBT, Sigma Fast@ tablets). Molecular weight of proteins was estimated with a mixture of protein standards: myosin (202 kDa), b-galactosidase (109 kDa), bovine serum albumin (78 kDa), and ovalbumin (46.7 kDa). Binding of the antibodies to barley AO proteins was detected by immunoprecipitation using protein-A-Sepharose CL-4B (Pharmacia). All of the activity (detected after native-PAGE) in the samples was removed when the antibodies and Sepharose Aldehyde oxidases in barley 65 were added, while all of the activity remained in the supernatant when control mouse serum was used. Soluble proteins were estimated by the Bio-Rad micro assay modification of the Bradford procedure (1976) using crystalline bovine serum albumin as a reference. MoCo determination Molybdenum cofactor activity in plant tissue was estimated using a pleiotropic nit-1 mutant of Neurospora crassa according to Nason et al. (1970). The original procedure (Mendel et al., 1985) was carried out with recent modifications (Sagi et al., 1997; Savidov et al., 1997b). Results Differences between AO proteins extracted from plant organs AO proteins were detected following gel electrophoresis of extracts of roots, leaves and seeds of barley. At least four bands were observed, three of them (AO-2, AO-3, AO-4) in roots, with marked differences in mobility during gel electrophoresis and subsequent staining with indole-3-aldehyde as substrate (Fig. 1). Additional aldehydes used were benzaldehyde, acetaldehyde and heptaldehyde. The AO proteins showed different affinity for the substrates used. Enzyme activity was most prominent with indole-3-aldehyde as substrate and differences in intensity of AO bands were not significant. Two bands (AO-3 and AO-4) were detected in gels with benzaldehyde, acetaldehyde and heptaldehyde as substrates ( Fig. 1B). The AO-4 band, with the highest mobility in the gel, showed its strongest affinity for heptaldehyde, acetaldehyde and benzaldehyde. The AO-1 band in roots could be observed with benzaldehyde, although its activity was very low (Fig. 1B). A single AO band (AO-1) was determined in leaves using each of the substrates although it reacted best with indole-3-aldehyde. High AO activity in seeds was detected with heptaldehyde, indole3-aldehyde and benzaldehyde. Acetaldehyde was a poor substrate for seed AO (not shown). Immunological analysis Polyclonal mouse antibodies raised against maize AO ( Koshiba et al., 1996) cross-reacted with at least three proteins in roots and a fourth band in leaves and seeds after native PAGE (Fig. 2A). These proteins corresponded to the location of AO proteins as revealed by their staining with aldehydes in gels after native PAGE ( Fig. 1A). The differences in molecular weight between the AO proteins extracted from roots, leaves and seeds persisted following exposure to SDS-PAGE and immunoblotting. Two polypeptides with molecular weights of about 160 kDa and 145 kDa each were detected in roots ( Fig. 2B). Only one protein band appeared after immunoblotting of leaf AO, with a molecular weight of 150 kDa. Immunoblotting of seed extracts yielded two polypeptides with molecular masses of 150 kDa and 85 kDa each after SDS-PAGE. The 85 kDa polypeptide may be either a degradation piece of the 150 kDa subunit produced by the extraction procedure or a latent unused precursor. A polypeptide with similar molecular weight has been observed previously in maize coleoptiles ( Koshiba et al., 1996) and tomato leaves (Ori et al., 1997). Other properties of AO in barley roots, leaves and seeds Root AO lost almost all activity when heated at 80 °C for 2.5 min prior to native PAGE (Fig. 3). Leaf and seed AO remained active after heat pretreatment of the extract at 85 °C for 2.5 min before native PAGE ( Fig. 3). The highest activity of leaf AO was detected with samples preheated between 65 and 80 °C. Menadione, a potent inhibitor of animal AO did not affect enzyme activity of roots and leaves of plants (data not shown) confirming the lack of inhibition previously reported for AO of maize coleoptiles ( Koshiba et al., 1996). Effect of tungstate on root and leaf AO activity Fig. 1. Native PAGE of AO proteins in barley. (A), AO isoforms in barley roots, leaves and seeds detected after native PAGE and developed with indole-3-aldehyde as substrate. (B) AO protein bands of barley roots developed with: indole-3-aldehyde (1), benzaldehyde (2), acetaldehyde (3), and heptaldehyde (4). Amount of proteins loaded per each lane: 230 mg for leaves, 230 mg for roots and 259 mg for seeds. Addition of 250 mM Na WO to the growth medium 2 4 inhibited AO activity in roots and leaves of 6-d-old seedlings. Tungstate inhibition of AO in roots was more prominent than in leaves ( Figs 4, 5). Na MoO (250 mM ) 2 4 66 Omarov et al. Fig. 2. Western blot analysis of AO proteins in barley organs. (A) Immunoblot made after native PAGE of extracts of leaves, roots and seeds of barley. (B) Immunoblot following SDS-PAGE of extracts of leaves, roots and seeds of barley. Arrows indicate molecular weight of AO polypeptides and standard protein markers. Fig. 3. Native PAGE of AO proteins of barley roots, leaves and seeds following heat pre-treatment prior to electrophoresis. Extract samples were heated in a water bath at different temperatures during 2.5 min followed by immediate cooling in ice. AO activity was determined with indole-3-aldehyde as substrate. Fig. 5. AO activity in roots of barley seedlings as affected by tungstate and molybdate. AO protein bands were developed with acetaldehyde and indole-3-aldehyde as substrates. 110 mg of proteins were loaded per each lane. Discussion Fig. 4. AO activity in leaves of barley seedlings as affected by tungstate and molybdate. AO activity was developed with indole-3-aldehyde as substrate. 230 mg of proteins were loaded per each lane. did not increase AO activity very much either in the leaves or the roots of seedlings. Tungstate also decreased MoCo activity in leaves (Fig. 6) and roots of seedlings (data not shown), although to a lower extent than AO. Several isoforms of AO in potato tubers (Rothe, 1974) and in maize coleoptiles ( Koshiba et al., 1996) have been observed. Recent observations have shown that staining of AO after native PAGE of Arabidopsis seedling extracts revealed the distribution of AO in an organ-specific manner. One AO-1 (with higher substrate preference for indole-3-aldehyde) was highly expressed in roots of both wild-type and sur 1 auxin-overproducing mutant plants and also specifically in the hypocotyl of the mutant seedlings, whereas AO-3 was detected in cotyledons exhibiting a marked substrate preference for naphthaldehyde (Seo et al., 1998). Four AO proteins were observed in barley plants, of Aldehyde oxidases in barley 67 Fig. 6. MoCo activity in leaves of barley seedlings as affected by tungstate. Bars indicate standard error of the mean. which at least three were detected in plant roots while only one major band was observed in leaves and seeds, capable of oxidizing to different degrees a number of aliphatic and aromatic aldehydes (Fig. 1). Western blotting revealed three of the AO proteins in roots ( Fig. 2A). Plant AO catalyses the oxidation of abscisic aldehyde (ABAld ) to ABA ( Walker-Simmons et al., 1989; Sindhu et al., 1990; Leydecker et al., 1995) and indole3-acetaldehyde (IAAld ) to IAA ( Koshiba et al., 1996). Are these multiple functions of a single enzyme protein? In view of the differences observed between leaf and root AO, the feasibility that similar but not identical proteins catalyse the oxidation of ABAld and IAAld should not be ignored. The main site of ABA synthesis is the root from where the hormone is transported to the shoot, especially under drought or saline conditions ( Wolf et al., 1990; Bano et al., 1993). ABA synthesis in roots was enhanced under salinity conditions (Schnapp et al., 1990), complementing recent observations on the enhancement of AO activity in barley roots by salinity and ammonium and the depression of AO activity by nitrate in the nutrient medium (Omarov et al., 1998). It is not completely clear yet how much of the increased levels of AO in roots of salt-stressed plants may be the result of enhanced expression of genes coding for the enzyme components (MoCo and AO apoprotein) or by posttranslational modulation of existing enzyme molecules as shown for the Mo-enzyme nitrate reductase ( Kaiser and Huber, 1996; Bachmann et al., 1996; Moorhead et al., 1996). AO proteins extracted from roots and leaves exhibited different mobility during native gel electrophoresis ( Fig. 1). Differences between AO proteins extracted from roots, leaves and seeds were also detected after SDSPAGE followed by Western blots. Two polypeptides with molecular weights of 160 kDa and 145 kDa were detected in roots ( Fig. 3), combinations (dimerization) of which may generate the four isomers observed in native PAGE of barley tissue extracts. Root, leaf and seed AO proteins differed in their susceptibility to high temperature pretreatments. Root AO was almost completely inactivated in extracts subjected to heating at 80 °C for 2.5 min while leaf AO was far more stable and showed even increased activity when pretreated at temperatures of 65–80 °C (Fig. 3). AO of maize coleoptiles lost all its activity after boiling for 5 min but was relatively stable at 60–70 °C ( Koshiba and Matsuyama, 1993). AO activity of cucumber seedlings was enhanced by heating enzyme preparations at 70 °C (Bower et al., 1978). The presence of AO in seeds (Figs 1A, 2) may be related to ABA production in the dormant embryo. AO in dry seeds of barley was localized almost exclusively in the embryo while the endosperm exhibited mere traces (not shown). Seed dormancy and ABA content of several plant species were very low in acid soils due to insufficient uptake of Mo, but it increased following Mo applications to the soil or the leaves (Modi and Cairns, 1994). In dry pea (Pisum sativum L.) seeds, exogenous sodium molybdate was absolutely required for MoCo determination ( Kildibekov et al., 1996). Therefore, the increase in ABA level in seeds following the application of exogenous Mo suggests the involvement of a MoCo-containing enzyme (presumably AO) in ABA synthesis and seed dormancy. Tungsten substituted for molybdenum in the MoCocontaining nitrate reductase inactivating the enzyme (Deng et al., 1989; Huber et al., 1994; Mendel, 1997). Xanthoxal, presumably the first free C molecule in the 15 ABA biosynthesis pathway (Milborrow et al., 1997; Lee and Milborrow, 1997), increased by 2.6-fold when AO activity was blocked by tungstate in avocado (Persea americana) fruits. The effect of tungstate on root and leaf barley AO proteins (Figs 4, 5) provides additional evidence of Mo involvement in the catalytic centres of the AO proteins observed in roots and leaves. AO inhibition by tungstate in roots of barley seedlings was more prominent than in leaves ( Figs 4, 5). This may indicate (a) differences in the biochemical properties of root and leaf AO proteins; (b) different rates of enzyme turnover which is required for the insertion of tungsten into pterin instead of Mo or (c) a limited transport of tungstate from roots to shoots. AO in plants consists of a group of several proteins with different electrophoretic mobility, substrate specificity and subunit composition. These subunits, which in different combinations give rise to the isozymes observed, may be the products of the cDNAs isolated from maize (Sekimoto et al., 1997), tomato (Ori et al., 1997) and 68 Omarov et al. Arabidopsis (Sekimoto et al., 1998). Present evidence does not allow at this stage to pinpoint which and how much each of the resulting dimers is involved in specific metabolic tasks in the plant organs studied. This diversity of AO proteins may be related to special metabolic roles in the plant organs and/or different locations within a given organ. Acknowledgements This work has been possible thanks to the financial support of AID/CDR to project CA15–124, the Fohs Foundation ( USA) and ICA (Israel ). Our gratitude to RR Mendel ( Technical University of Braunschweig, Germany) for valuable discussions. References Bachmann M, Shiraishi N, Campbell WH, Yoo BC, Harmon AC, Huber SC. 1996. Identification of Ser-453 as the major regulatory phosphorylation site in spinach leaf nitrate reductase. The Plant Cell 8, 505–517. Bano A, Dörffling K, Bettin D, Hahn H. 1993. Abscisic acid and cytokinins as possible root-to-shoot signals in xylem sap of rice plants in drying soil. Australian Journal of Plant Physiology 20, 109–115. Bower PJ, Brown HM, Purves WK. 1978. Cucumber seedling indole acetaldehyde oxidase. Plant Physiology 61, 107–110. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Deng M, Moureaux T, Caboche M. 1989. Tungstate, a molybdate analog inactivating nitrate reductase, deregulates the expression of the nitrate reductase structural gene. Plant Physiology 91, 304–309. Hoagland DR, Arnon DI. 1938. The water culture method for growing plants without soil. California Agricultural Experimental Station Circular 347, 1–32. Huber SC, Huber JL, Kaiser WM. 1994. Differential response of nitrate reductase and sucrose-phosphate synthaseactivation to inorganic and organic salts, in vitro and in situ. Physiologia Plantarum 92, 302–310. Kaiser WM, Huber SC. 1996. Correlation between apparent activation state of nitrate reductase (NR), NR hysteresis and degradation of NR protein. Journal of Experimental Botany 48, 1367–1374. Kildibekov NA, Omarov RT, Antipov AN, Shvetsov AA, Mironov EA, L’vov NP. 1996. Purification of a molybdo-cofactorcontaining protein from pea seeds and identification of molybdopterin. Plant Physiology and Biochemistry 34, 677–682. Koshiba T, Matsuyama H. 1993. An in vitro system of indole3-acetic acid formation from tryptophan in maize (Zea mays) coleoptile extracts. Plant Physiology 102, 1319–1324. Koshiba T, Saito E, Ono N, Yamamoto N, Sato M. 1996. Purification and properties of flavin- and molybdenumcontaining aldehyde oxidase from coleoptiles of maize. Plant Physiology 110, 781–789. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lee HS, Milborrow BV. 1997. Endogenous biosynthetic precursors of (+)-abscisic acid. V. Inhibition by tungstate and its removal by cinchonine shows that xanthoxal is oxidized by a molybdo-aldehyde oxidase. Australian Journal of Plant Physiology 24, 727–732. Leydecker MT, Moureaux T, Kraepiel Y, Schnorr K, Caboche M. 1995. Molybdenum cofactor mutants, specifically impaired in xanthine dehydrogenase activity and abscisic acid biosynthesis, simultaneously overexpress nitrate reductase. Plant Physiology 107, 1427–1431. Marin E, Marion-Poll A. 1997. Tomato flacca mutant is impaired in ABA aldehyde oxidase and xanthine dehydrogenase activities. Plant Physiology and Biochemistry 35, 369–372. Mendel RR. 1997. Molybdenum cofactor of higher plants: biosynthesis and molecular biology. Planta 203, 399–405. Mendel RR, Kirk DW, Wray JL. 1985. Assay of molybdenum cofactor of barley. Phytochemistry 24, 1631–1634. Milborrow BV, Burden RS, Taylor HF. 1997. The conversion of 2-cis-[14C ] xanthoxic acid into [14C ]ABA. Phytochemistry 45, 257–260. Modi AT, Cairns ALP. 1994. Molybdenum deficiency in wheat results in lower dormancy levels via reduced ABA. Seed Science Research 4, 329–333. Moorhead G, Douglas P, Morrice N, Scarabel M, Aitken A, MacKintosh C. 1996. Phosphorylated nitrate reductase from spinach leaves is inhibited by 14–3-3 proteins and activated by fusiococcin. Current Biology 6, 1104–1113. Miyata S, Suzuki Y, Kamisaka S, Masuda Y. 1981. Indole3-acetaldehyde oxidase of pea seedlings. Physiologia Plantarum 51, 402–406. Nason A, Antoine AD, Ketchum PA, Frazier WA, Lee DK. 1970. Formation of assimilatory nitrate reductase by in vitro intercistronic complementation in Neurospora crassa. Proceedings of the National Academy of Science, USA 65, 137–144. Omarov RT, Sagi M, Lips SH. 1998. Regulation of aldehyde oxidase and nitrate reductase in roots of barley (Hordeum vulgare L.) by nitrogen source and salinity. Journal of Experimental Botany 49, 897–902. Ori N, Eshed Y, Pinto P, Paran I, Zamir D, Fluhr R. 1997. TAO1, a representative of the molybdenum cofactor containing hydroxylases from tomato. Journal of Biological Chemistry 272, 1019–1025. Rajagopal R. 1971. Metabolism of indole-3-acetaldehyde. III. Some characteristics of aldehyde oxidase of Avena coleoptiles. Physiology Plantarum 24, 272–281. Rothe GM. 1974. Aldehyde oxidase isoenzymes (EC 1.2.3.1) in potato tubers (Solanum tuberosum). Plant Cell Physiology 15, 493–499. Sagi M, Omarov RT, Lips SH. 1998. Xanthine dehydrogenase, nitrate reductase and aldehyde oxidase in ryegrass as affected by nitrogen and salinity. Plant Science (in press). Sagi M, Savidov NA, L’vov NP, Lips SH. 1997. Nitrate reductase and molybdenum cofactor in annual ryegrass as affected by salinity and nitrogen source. Physiologia Plantarum 99, 546–553. Savidov NA, L’vov NP, Sagi M, Lips SH. 1997a. Molybdenum cofactor biosynthesis in two barley (Hordeum vulgare L.) genotypes as affected by nitrate in the tissue and in the growth medium. Plant Science 122, 51–59. Savidov NA, Sagi M, Lips SH. 1997b. The assay of the molybdenum cofactor in higher plants as affected by pyridine nucleotides and nitrate. Plant Physiology and Biochemistry 35, 419–426. Sekimoto H, Seo M, Dohmae N, Takio K, Kamiya Y, Koshiba T. 1997. Cloning and molecular characterization of plant Aldehyde oxidases in barley 69 aldehyde oxidase. The Journal of Biological Chemistry 272, 15280–15285. Sekimoto H, Seo M, Kawakami N, Komano T, Desloire S, Liotenberg S, Annie Marion-Poll, Caboche M, Kamiya Y, Koshiba T. 1998. Molecular cloning and characterization of aldehyde oxidases in Arabidopsis thaliana. Plant and Cell Physiology 98, 433–442. Seo M, Akaba Sh, Oritani T, Delarue M, Bellini C, Caboche M, Koshiba T. 1998. Higher activity of an aldehyde oxidase in the auxin-overproducing superroot1 mutant of Arabidopsis thaliana. Plant Physiology 116, 687–693. Sindhu RK, Griffin DH, Walton DC. 1990. Abscisic aldehyde is an intermediate in the enzymatic conversion of xanthoxin to abscisic acid in Phaseolus vulgaris L. leaves. Plant Physiology 93, 689–694. Schnapp S, Bresson R, Hasegawa P. 1990. Carbon use efficiency and cell expansion of NaCl-adapted tobacco cells. Plant Physiology 93, 384–388. Schwartz SH, Léon-Kloosterziel KM, Koornneef M, Zeevaart JAD. 1997. Biochemical characterization of the aba2 and aba3 mutants in Arabidopsis thaliana. Plant Physiology 114, 161–166. Taylor IB, Linforth RST, Al-Naieb RJ, Bowman WR, Marples BA. 1988. The wilty tomato mutants flacca and sitiens are impaired in the oxidation of ABA-aldehyde to ABA. Plant, Cell and Environment 11, 739–745. Walker-Simmons M, Kudra DA, Warner RL. 1989. Reduced accumulation of ABA during water stress in a molybdenum cofactor mutant of barley. Plant Physiology 90, 728–733. Winer L, Riov J, Goren R. 1993. Catabolism of indole-3-acetic acid in citrus leaves: identification and characterization of indole-3-aldehyde oxidase. Physiologia Plantarum 89, 220–226. Wolf O, Jeschke WD, Hartung W. 1990. Long distance transport of abscisic acid in NaCl-treated intact plants of Lupinus albus. Journal of Experimental Botany 41, 593–600.