Plant Cell Physiol. 38(3): 268-273 (1997) JSPP © 1997 Conversion of Indole-3-Acetaldehyde to Indole-3-Acetic Acid in Cell-Wall Fraction of Barley {Hordeum vulgare) Seedlings Ken-ichi Tsurusaki1, Kazuyoshi Takeda2 and Naoki Sakurai3 1 Faculty of Liberal Arts, Fukuyama University, Fukuyama, 729-02 Japan 2 Research Institute for Bioresources, Okayama University, Kurashiki, Okayama, 710 Japan 3 Department of Environmental Studies, Faculty of Integrated Arts & Sciences, Hiroshima University, Higashi-Hiroshima, 739 Japan The cell-wall fraction of barley seedlings was able (Trp) has been suggested as a primary precursor of IAA to oxidize indole-3-acetaldehyde (IAAld) to form IAA, (Gordon 1954, Gibson et al. 1972, Monteiro et al. 1988, whereas the fraction did not catalyze the conversion of in- Cooney and Nonhebel 1991, Bialek et al. 1992, Koshiba dole-3-acetonitrile or indole-3-acetamide to IAA. The activ- and Matsuyama 1993, Koshiba et al. 1995), because Trp iDownloaded from https://academic.oup.com/pcp/article/38/3/268/1928462 by guest on 24 September 2021 s ity was lower in a semi-dwarf mutant that had an endog- similar in structure to IAA and is ubiquitous in plant enous IAA level lower than that of the normal isogenic tissues. strain [Inouhe et al. (1982) Plant Cell Physiol. 23: 689]. Two pathways of IAA biosynthesis from L-Trp have The soluble fraction also contained some activity; the activ- been proposed in higher plants: Trp —• indole-3-pyruvic ity was similar in the normal and mutant strains. The op- acid -»indole-3-acetaldehyde (IAAld) ->• IAA; or Trp -> timal pH for the conversion of IAAld to IAA in the cell- tryptamine —• IAAld -* IAA. With the unequivocal dem- wall fraction was 7; that of soluble fraction was 6. The Km onstration of indole-3-acetaldehyde oxime, which appears value of the cell-wall fraction for IAAld was 5 fM; that of to be synthesized from N-hydroxy-tryptophan, in some soluble fraction was 31 fiM. The activity was not solubi- higher plants (Ludwig-Miiler and Hilgenberg 1988) and lized by treatments with 1% Nonidet P-40,1M NaCI, 3 M its onward metabolism to IAA in several higher plants LiCl, or 50 mM MgCl2. The oxidation activity was in- (Rajagopal et al. 1993), two other routes became apparent: creased by the addition of NAD+. These results suggest indole-3-acetaldehyde oxime -•• IAAld -»• IAA; and in- that IAAld oxidation activity is bound to cell-wall com- dole-3-acetaldehyde oxime —• indole-3-acetonitrile (IAN) —• ponents and that the lower level of IAA in the mutant prob- IAA. Indole-3-acetamide (IAM) has been reported as an im- ably results from reduced activity of oxidation enzyme portant precursor of IAA in some higher plants (Kawa- bound to cell-wall components. guchi et al. 1991, 1993, Saotome et al. 1993, Rajagopal et al. 1994). On the other hand, IAA may not be directly syn- Key words: Barley — Biosynthesis — Hordeum vulgare — thesized from Trp but from anthranilate or indole, which IAA — Indole-3-acetaldehyde — Indole-3-ethanol. are precursors of Trp (Baldi et al. 1991, Wright et al. 1991, Michalczuk et al. 1992). The question of IAA synthesis involves not only the primary precursor but also the direct precursor of IAA. Most physiological studies of the cause of dwarfism in IAAld (Rajagopal 1967a, b, 1968, 1971, Bower et al. 1978, plants have focused on the role of gibberellins (Brian and Purves and Brown 1978, Miyata et al. 1981, Koshiba et al. Hemming 1955, Lockhart 1956, Cooper 1958, Loy and Liu 1996), IAN (Normanly et al. 1993, Bartel and Fink 1994, 1974, Perez et al. 1974). However, dwarfism has also been Bartling et al. 1994) and IAM (Kawaguchi et al. 1991,1993, attributed to other causes (Kuraishi 1974, Wylie and Ryugo Saotome et al. 1993, Rajagopal et al. 1994) have been sug- 1971). Van Overbeek (1938) first showed that lowered aux- gested as direct precursors, but the existence and amounts in content in dwarf corn seedlings is one of the causes of these compounds and their substrate specificities are still of dwarfism. Inouhe et al. (1982) demonstrated that the controversial. dwarfism of etiolated seedlings of eleven barley isogenic Recently, a possible role for aldehyde oxidase in IAA strains (Hordeum vulgare L. cv. Akashinriki) is primarily biosynthesis was suggested in maize coleoptile (Koshiba et caused by low levels of endogenous IAA in these strains. al. 1996). Aldehyde oxidase could catalyze the oxidation of The pathway of IAA biosynthesis has been extensively IAAld to form IAA (Rajagopal 1971, Bower et al. 1978, studied, but has not yet been demonstrated unequivocally Miyata et al. 1981). Koshiba and Matsuyama (1993) report- (Bandurski et al. 1995, Normanly et al. 1995). Tryptophan ed that an in vitro system of maize coleoptile extracts cata- lyzed the production of IAA from Trp and that the IAA- Abbreviations: CWF, cell-wall fraction; GC-(SIM-)MS, gas forming activity was co-purified with an IAAld oxidase. chromatography-(selected ion monitoring-) mass spectrometry; IAAld, indole-3-acetaldehyde; IPA, indole-3-propionic acid; IEt, However, the actual function of IAAld oxidase in IAA bio- indole-3-ethanol; IAM, indole-3-acetamide; IAN, indole-3-aceto- synthesis is not yet known. nitrile; SEF, soluble enzyme fraction; Trp, tryptophan. To determine the cause of the lower level of endoge- 268 IAA synthesis activity bound to cell walls 269 nous IAA in dwarf barley (Inouhe et al. 1982), the present of sugar, 3 mM ATP, and 3 mM MgSO4 in homogenizing medium c experiments were undertaken to investigate the contri- and was kept for 2 min at 30 C. The reaction was stopped by the bution of IAAld to the IAA biosynthesis pathway in the addition of 300 ftl of 20% SDS. The mixture was added to 4 ml of a mixture of 10 mM ammonium molybdate : 2.5 M sulfuric acid : normal and isogenic dwarf mutant of barley. We demon- acetone (1:1:2, v/v/v), then was mixed with 0.4 ml of 1 M citric strated that the insoluble cell-wall fraction exhibited sub- acid. The color intensity was measured at 355 nm. stantial IAAld oxidation activity which converted IAAld to HPLC—Separation of the products was achieved on an Inert - IAA and revealed that the activity of this cell-wall fraction sil ODS-2 column (4.6x 150mm, GL Sciences Inc., Tokyo), was lower in the dwarf barley strain, which had been previ- eluted isocratically with 20% acetonitrile (pH 5.0 adjusted with 20 mM triethylamine-acetic acid) at a flow rate of 0.8 ml min"1. ously shown to be contain lower levels of endogenous IAA The HPLC system consisted of a pump unit (model LC-6A, than the normal strain. Shimadzu Inc., Kyoto) connected to a system controller (model Downloaded from https://academic.oup.com/pcp/article/38/3/268/1928462 by guest on 24 September 2021 SCL-6A, Shimadzu Inc.). IAA and IEt were detected with an ex- citation at 280 ±5 nm and an emission at 350+5 nm by spectroflu- Materials and Methods orometry with a fluorometric detector (FP-200, Japan Spectro- Plant materials—Two isogeneic strains (uzu or semi-dwarf scopic CO., Tokyo). The IAA and IEt contents were calculated and its corresponding normal line) of barley (Hordeum vulgare from the ratio of the peak area of IPA to that of IAA or IEt, as de- L. cv. Akashinriki) were harvested at an experimental farm scribed by Kuraishi et al. (1989). of Research Institute for Bioresources, Okayama University, in GC-MS—Following HPLC, the fractions corresponding to 1993. About 700-800 seeds were soaked for 8 h in water and then IAA or IEt retention time were pooled and evaporated to dryness. germinated in the dark in plastic boxes (34 cm x 23 cm x 4 cm) fill- The dried samples were trimethylsilylated with a mixture of bis-tri- ed with moistened vermiculite at 25.5±0.5°C for 3 d. Coleoptiles, methylsilyl trifiuoroacetamide (BSTFA, Tokyo Kasei Kogyo Co., including first leaves, selected for uniformity of seedling length Tokyo) and acetonitrile (1 : 1, v/v) at 70° C for 1 h. The trimethyl- (15-30 mm for the dwarf and 30-50 mm for the normal strain) silylated sample was characterized by GC-MS or GC-SIM-MS of were weighed. The material was immediately used for experiments QP-1000 (Shimadzu Inc.). Ionization of the compound was car- or frozen at — 80°C until use. ried out by electron impact at 70 eV. The gas chromatograph was Enzyme fractionation—All manipulations were performed at equipped with a 30 m fused silica capillary column (CBJ 17, 2-4°C. One hundred samples (normal, ca. 3.5 g in fresh weight; Shimadzu Inc.). uzu, ca. 2.4 g in fresh weight) were homogenized by mortar and pestle in 4 to 8 ml of 50 mM Tris-HCl (pH 7.2) containing 5 mM Results Na-EDTA, 1 mM dithiothreitol, 10 mM MgCl2, 1 mM benzami- dine, and 0.1 mM 4-(2-aminoethyl) benzensulfonyl fluoride. The homogenate was centrifuged at 1,000 xg for 20 min at 4°C. The Comparison of the IAA synthesis activity of SEF and supernatant was designated as soluble enzyme fraction (SEF). The CfFF—Table 1 shows the IAA synthesis activity from pellet was suspended in homogenizing medium with \% Nonidet different precursors by SEF or CWF of the normal and uzu P-40 and 1 M NaCl and homogenized again. The homogenate was strains. When the activity is expressed per seedling, SEF centrifuged at 1,000 x g for 20 min, washed 3 times with the ho- converted IAAld to IAA more than did CWF in both the mogenizing medium, and suspended in the same medium (4 to 8 ml).