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Effect of Light on Growth and Metabolic Activities in Pea Seedlings II. Changes in the IAA Content and Activities of Enzymes Involved in the IAA Biosynthesis during Growth
Biochem. Physiol. Pflanzen 176, 35 -43 (1981)
Effect of Light on Growth and Metabolic Activities in Pea Seedlings II. Changes in the IAA Content and Activities of Enzymes Involved in the IAA Biosynthesis during Growth Y. SUZUKI,
S.
KAMISAKA,
H.
YANAGISAWA,
S. MIYATA and
Y. MASUDA
Department of Biology, Faculty of Science, Osaka City University, Osaka, Japan Key Term Index: epicotyls, IAA, tryptophan aminotransferase, indolepyruvic acid decarboxylase, indoleaceta.ldehyde oxidase, amine (tryptamine) oxidase, light inhibition; P1:sum sativum.
Summary 1'he effect of light and dark conditions on the IAA content and the activities of tryptophan aminotransferase, indolepyruvic acid decarboxylase, indoleacetaldehyde oxidase and amine oxidase in pea (Pisum sativum L., cv. Alaska) seedlings was studied and the following results were obtained. 1. The endogenous IAA content was higher in shoots of dark-grown pea seedlings than in those of light-grown ones. When seedlings which had been grown in the dark were transferred to light, the endogenous IAA content decreased. When, on the other hand, the light-grown seedlings were transferred to darkness, the IAA content increased. 2. No significant difference in the effect of the light and dark conditions was found on the activities of tryptophan aminotransferase, indolepyruvic acid decarboxylase and indoleacetaldehyde oxidase in the epicotyl. 3. The activity in the apical part of amine oxidase which catalyzes the conversion of tryptamine to indoleacetaldehyde was inhibited considerably under light conditions. These results indieate that the increased IAA content in the dark conditions is not likely to depend on specific regulatory mechanisms at the enzyme level. Although the amine oxidase activity was found to be high in the dark-grown pea seedlings, the function of this enzyme in IAA biosynthesis remains unsolved.
Introduction
Growth and development in higher plants are under the genetic control and also under the environmental control, the former being often regulated by the latter, such as light conditions (cf. SACHS 1872). In our previous paper (MASUDA et al. 1981) we reported that the mechanical property of the epidermal cell wall and the extension potential of the inner tissue of the internodes are remarkably influenced by light conditions. We also found that the metabolic turnover of sugars of cell wall polysaccharides, particularly of neutral sugars in the pectic fraction, is closely related with the extension of the internodes as affected by light conditions (NISHITANI and MASUDA 1979). Plant growth substances play roles as effectors in the regulation of growth and development. Among the substances, auxin is believed to playa leading role in the reguAbbreviations: IAA, indole-8-acetic acid; EDTA, sodium ethylenediamine tetraacetate; IAAld, indoleacetaldehyde; IPA, B-indolepyruvic acid; TPP, thiamine pyrophosphate; TCA, trichoroacetic acid; PMS. phenazine mcthosulphate.
36
Y.
SUZUKI
et al.
lation (THIMANN 1972). However, the correlation of intact growth in plants with auxin metabolism, synthesis and degradation under different environmental conditions has not been well established. Thus, we decided to investigate changes in the IAA content and in the activities of different enzymes involved in the IAA biosynthesis in pea seedlings grown in darkness and in light. This study tries to correlate shoot growth under different light conditions with auxin metabolism. Material and Methods Plant material Pea (Pisum sativum L., cv. Alaska) seeds were sown and germinated in moist vermiculite in plastic trays at 25 00. The trays were kept under continuous light (ca. 3,000 lux at plant level) or in total darkness. The seedlings in another tray were first grown in the dark for 5 days and then transferred to light and kept for 2 days, or they were first grown in the light then transferred to darkness.
Preparation of enzyme frtwtions 1. Tryptophlm aml~notransftrase: The exeised shoots, surface sterilized for 10 min in 1 % benzalkonium chloride solution and then washed thoroughly with deionized water, were homogenized in a mortar with three volumes (vjw) of cold 0.1 M phosphate buffer (pH 7) containing 10 mM 2-mercaptoethanol, 1 mM EDTA, 10 l,M pyridoxal phosphate and wet Polyclar AT (shoots: Polyclar AT = 1: 0.1 in w/w). The resulting slurry was passed through a layer of cotton cloth and then centrifuged for 20 min at 10,000' g. The supernatant was fraetionated with ammonium sulfate, and the precipitate obtained with 80 % saturation was dissolved in a minimal amount of 50 roM phosphate buffer (pH 8) containing 10 mM 2-mercaptoethanol, 1 mM EDT A and lO.uM pyridoxal phosphate. After centrifugation for 30 min at 10,000 . g, the supernatant was passed through a column (2.2 x 65 cm) of Sephadex G-25, then the protein solntion eluted was used for the enzyme assay. 2. IP.!! decarboxylase: The excised shoots were homogenized in a Waring blender with twice volumes (v/w) of 0.1 M phosphate buffer (pH 7) containing 1 roM cysteine HOI, 0.1 roM EDTA, 0.1 mM TPP, 1 mM MgS0 4, 20% (vjv) glycerol and PolycIar AT (shoots: Polyclar AT = 1: 0.06 in wjw). The homogenate was filtered through a layer of cotton cloth and centrifuged for 30 min at 10,000· g. The superna.tant was fractionated with ammonium sulfate and the precipitate obtained at 80 % saturation was dissolved in a minimum amount of 50 mM phosphate buffer (pH 7) containing 10- :>.M TPP and 1 mM MgS04 and then dialyzed overnight against 1,000 ml of the same buffer solution with three changes. The dialysate was used for the enzyme assay. 3. I AAld oxidase: The enzyme was prepared from the excised shoots and the procedure was the same as those of IPA decarboxylase preparation, except that TPP and MgS04 were not included in the extraction medium. 4. Amine o:cidase: The excised shoots were homogenized in a mortar with twice volumes (v/w) of cold 0.1 M phosphate buffer (pH 7). The homogenate was filtered through a layer of cotton cloth a,nd centrifuged for 20 min at 10,000· g. The supernatant was used for the enzyme assay.
Determination of endogenous IAA. IAA in pea shoots was extracted with methanol. The purification of IAA in the methanol extract was carried out as deseribed earlier (KAMISAKA and LARSEN 1977). 1-14C-IAA (3,000 dpm) was added to the methanol extract to correct IAA loss during purification, then the methanol fraction was evaporated under reduced pressure at 40 °0. To the residue, 10 ml of 0.5 M K2 HPO, solution was added. Lipoidal substances were extracted three times with light petrolium ether and twic.e with ethyl ether. Then, the pH of the solution was adjusted to 3 with 2.8 M Hg P04 solution. The acidic fraction was shaken with ethyl ether. The ethyl ether fraction was a.gain shaken with 10 ml
IAA Content and IAA Biosynthesis Enzymes
37
of 0.05 M KzHP0 4 solution. After adjusting the pH of the 0.05 M KzHP04 fraction to 3 with 0.28 M H3 P0 4 solution, the acidic ethyl ether fraction was prepared by sha,king the KzHP0 4 fraction with ethyl ether. The determination of IAA in the acidic ether fraction was carried out by the modified indolo-{X-pyrone fluorescence method of KAMISAKA and LARSEN (1977).
Enzyme assays 1. Tryptophan aminotransferase: For the estimation of tryptophan aminotransferase activity, indolepyruvate-borate complex, the product of the enzyme action in the presence of borax-HCI buffer was assayed spectrophotometrically according to the method of TRUELSEN (1972) and that of MATHERON and MOORE (1973). The routine reaction mixtures consisted of 0.5 ml of enzyme preparation, 1 ml of 0.5 M borax-HCl buffer solution (pH 8.5), 0.4 ml of 0.1 M tryptophan (in 0.2 N HCI), 0.1 ml of 0.2 M {X-ketoglutarate (pH 6.5) and 0.1 ml of 1 mM pyridoxal phosphate solution. Total volume was adjusted to 2.5 ml with deionized water. After reaction mixtures were incubated for 60 min at 30 °c with shaking, the reaction was stopped by the addition of 0.5 ml of 24 % TCA. The mixtures were then centrifuged for 10 min at 3,000 rpm. Two ml of each supernatant was added to 2 ml of 1 M sodium arsenate-boric acid mixture (pH 6.5) separately and allowed to stand for 20 min. Then the absorbance was measured at 328 nm. Reaction mixtures minus {X-ketoglutarate were used as controls. 2. J P A decarboxylase activity: For the estimation of IP A decarboxylase activity, the evolution of carbon dioxide caused by IPA in the presence of enzyme was assayed manometrically at 30°C. The reaction mixture consisted of 2 ml of enzyme solution, 0.3 ml of 10 mM TPP and 10 mM IP A (adjusted to pH 7 with 1 M phosphate buffer) in the main chamber. Side-arm contained 0.2 ml of 2 N H 2 SOt which was transferred to the main chamber at the end of the experiment. Control was used without the addition of IPA. The center well contained 0.2 ml of 15 % KOH or 2 N HzSO.1• The data were calculated from the carbon dioxide evolution after 2 h. 3. J A..4~d oxidase: The enzyme activity was estimated by determining the oxygen upta.ke from air in a Wa,rburg flask at 30°C. The reaction mixture was composed of 1 ml of 0.1 M phosphate buffer (pH 7), 1 ml of enzyme solution and 0.3 ml of 20 mM IAAld bisulphite (side-arm). Control was used without the addition of substrate. The center well contained 0.1 ml of 15% KOH. The data were cahmlated from the oxygen uptake in the first 20 min. 4. Aml:ne oX1:dase: Amine oxida.se activity for tryptamine and putrescine was estimated using a. Clark oxygen electrode at 30°C and the consumption of soluble oxygen was measured with a. TOA polyrecorder (Model 10 A) (SUZUKI and YANAGISAWA 1976). The standard assay system was as follows: 0.4 ml of enzyme preparation, 1 mlof 0.1 M phosphate buffer (pH 8), 0.1 ml of beef liver crystalline catalase (250 p.gjO.l ml) and 0.5 ml of tryptamine· HCl or putrescine· 2 HCl (50 mM each). Total volume was adjusted to 3.2 ml with deionized water. Colorimetric estimation of the enzyme activity for putreseine was also assayed by a method similar to that described by HOLMSTED'f et aJ. (1961). The reaction mixture consisted of 0.5 ml of 50 mM putrescine· 2 HCl solution, 0.1 ml of 0.1 M phosphate buffer (pH 7), 0.2 m] of enzyme preparation, 0.2 rul of 0.1 % o-aminobenzaldehyde solution (in ethanol) and 0.1 m] crysta11ine beef liver catalase (250 !lg/0.1 ml). Total volume was adjusted to 2.6 ml with deionized water. The mixture was incubated for 5 min at 30°C with shaking, and the reaction was stopped by adding 1 ml of 50 % TCA, then 4 ml of deionized water was added. The mixture was filtered off after standing for 30 min in an ice box, then the absorbance of the filtrate was estimated at 435 nm. Both reactions were started by the addition of enzyme preparations.
Protein determination The protein content was determined by the method of LOWRY et a1. (1951) using bovine serum albumin as the standard.
38
Y.
SUZUl\l
et al.
Results
Effect of dark and light on the IAA content in pea seedlings The effects of different regimes of the dark and light on the IAA content in pea shoots were determined as shown in Table 1. When the dark-grown pea seedlings were exposed to light, the IAA content per fresh weight or per shoot usually was lower than that of the seedlings grown in the dark for 5 + 2 days. However, if the reverse order of the light treatment was used, the IAA content increased in the dark, suggesting that the dark condition stimulated the synthesis of IAA or prevented its decomposition. In contrast, light might induce the conditions of IAA destruction or inhibition of IAA synthesis. Table 1. The endogenous I AA content in Alaska pea shoots grown in the dark and light. Pea seedlings were grown ill the dark and light and shoots were subjected to the IAA determinatioH. D5: grown for 5 days in the dark, D2: grown for 2 days in the dark, L2: grown for 2 days in the light L8: grown for 8 days in the light. Experiment
IAA content
Growth conditions
ng/g fresh weight
I
II
8.4 ± 0.6 14.6 ± 0.7 10.9 ± 1.8 32.3 ± 2.1 42.5 ± 1.5
D5 D5 ~ D2 D5 ~ L2 18 L8 ~ D2 L8 -,.. 1.2
30.9
±
2.0
ng/shoot
1.7
±
0.1 5.1 ± 0.2 2.6 ± 0.4 6.5 ± 0.4
11.9 8.1
± 0.4
-+:
0.5
Activities of tryptophan aminotransferase, IP A decarboxylase and IAAld oxidase in the dark- and light-grown pea seedlings Extracts from apical parts of dark- and light-grown pea seedlings were examined for the activity of tryptophan aminotransferase, IP A decarboxylase and IAAld oxidase. As shown in Table 2, no significant difference in the both conditions of light and dark was found for the activity of these enzymes in the extract of apical segments. Table 2. Activities of tryptophan aminotransferase, IP A decarboxylase and IAAld oxidase in Alaska
pea epicotyls grown in the dark and light. Pea seedlings were grown in the dark and light for 5 to 6 days or 7 to 8 days, and epicotyls were subjected to enzyme activity determination. Epicotyl length em
Growth conditions
7.4 ± 0.6 19.8 ± 2.5 7.2 ± 0.5
L7-8 D7-8 D5-6
Enzyme activities Tryptophan aminotransfera,se IPA nmoles/hr(mg protein
IP A decarboxylase IAAld oxidase ,ul CO 2(hrjmg ,Ill 02/hr/mg protein protein
10
0.16 0.11
13 15
4.56
5.10
39
IAA Content and IAA Biosynthesis Enzymes
Effect of light on amine oxidase (tryptamine oxidase) activity in the dark-grown pea seedlings Contrary to the above enzymes, the activity of tryptamine oxidase in the darkgrown pea epicotyls was five-fold as compared with that in light-grown ones, if the comparison was made on the same length of the epicotyl (Table 3). When the seedlings which had been grown for 5 days in the dark were transferred to light and kept for 2 days, the amine oxidase activity in the epicotyl was significantly decreased. However, such trend was not observed for tryptophan aminotransferase activity (Table 4). Table 3. Activity of amine oxidase in Alaska pea seedlings grown in the dark and light. Pea seedlings were grown for [) or 8 days in the dark and light and then epicot.yls were subjected to the enzyme activity determination. Epicotyl length (cm)
7.2
± 0.5
7.4. ± 0.6 2.9 :i-: 0.5
Growth ronditions
D5 L8 L5
Sped fie activity (nmole 02/min!mg protein) Tryptamine
Putrescine
21 4
188 22
12
41
Table 4. Activities of amine oxidase and tryptophan aminotransferase in Alaska pea seedlings grown
in the dark and light. Pea seedlings were grown for 5 or fnrther for 2 days in the dark and light and then epicotyls were subjected to enzyme activity determinations. Specific activities Epicotyl length cm
7.2 ± 0.5 19.8 ± 2.5 10.6 ± 1.7 2.9 ± 0.5 7.4±O.6 9.8 ± 0.8
Growth ('onditions
Amine oxidase (nmole 0z/ minjmg protein)
Tryptophan aminotra,nsferase (IPA nmolesjhrjmg
Tryptamine
Putrescine
protein)
D5
21
188
])5 --+ D2
23
252
I
113
12
41
:J
9'> '"'
15.3 13.3 13.5 10.1 10.0 13.0
Db --+ L2 15 I~5 --+ 13 15 -~ D2
,..,
U
-
67
Effect of dark and light on the distribution of amine oxidase activity in different shoot segments of pea seedlings In order to examine the distribution of amine oxidase activity in different shoot segments, different parts of the pea seedlings grown in the dark and light were excised
40
Y.
SUZUKI
et al.
Table 5. Activity of amine oxidase in Alaska pea seedl1:ngs grown in thp. dark and liyht. Pea seedlings were grown for 5 or 8 days in the dark and light a,nd ep:ieotyls were subjected to the enzyme activity determinations. A: apical part, M: middle part, B: basal part Epicotyl length em
7.2
±
0.5
10.6± 1.7 7.4 ± 0.6
Growth conditions
D5 D5 .- L2 L8
Enzyme a.ctivity Tryptamine (nmoles 02/minjmg protein)
Putrescine (nmoles Ll-pyrroline/ min/mg protein)
A
M
B
A
M
B
25 23 13
99 -~
19
19 4
6
425 425 285
285 301 65
81 48
4
27
and then separately analyzed. As shown in Table 5, the highest enzyme activity was found in the apical parts of the internode in the both conditions of dark and light. When the seedlings which had been grown in the dark for 5 days were transferred to the light, the ability of tryptamine oxidation in the basal part of the internode was significantly depressed as compared with the apical and middle parts of the internodes. In our experiments, the depression of amine oxidase activity was fairly large in the case of tryptamine oxidation rather than that of putrescine when the plant grown in the dark was exposed to the light (Table 5).
Discussion
We have found in the present study that there is a good correlation between the IAA content in the extract of epicotyl (Table 1) and epicotyl elongation (Table 2). The amount of 1AA in the dark-grown epicotyl as compared with that of light-grown one is relatively high. The reversible inhibition of the internode elongation of pea seedlings by light was also well correlated with the changes in the 1AA content (Table 1). Therefore, it appears that elongation of the epicotyl of pea seedlings is controlled by light via the control of 1AA content. On the other hand, the biosynthesis of 1AA from tryptophan has been considered to be carried out mainly via two different pathways, i.e. tryptophan -* indolepyruvic acid -* indoleacetaldehyde -* 1AA (first pathway) and tryptophan -* tryptamine -+ indoleacetaldehyde -+ 1AA (second pathway) (SHELDRAKE 1973). The pathways of 1AA biosynthesis in mung bean, barley and tomato have been studied extensively by Wightman and his co-workers (SCHN'EIDER et al. 1972; GIBSON et al. 1972a, b). They partially purified tryptophan aminotransferase and tryptophan decarboxylase from tomato shoots. However, the correlation of intact growth with auxin metabolism, synthesis and degradation under different environmental conditions such as light and darkness has not been studied. If the 1AA content depends on the rate of 1AA biosynthesis, we might expect that some enzyme activities related
IAA Content and IAA Biosynthesis Enzymes
41
to IAA biosynthesis in dark-grown pea seedlings differ from those grown in the light. As shown in Table 2, no significant difference in the both conditions of light and dark was found for the activity of three key enzymes involved in the first pathway of IAA biosynthesis, namely tryptophan aminotransferase, IPA decarboxylase and IAAld oxidase. TRUELSEN (1973) has shown that tryptophan aminotransferase activity in pea shoot tips which were grown in the light is about two-fold as compared with those grown in the dark. We have also found that in the pea seedlings light tends to increase the activity of tryptophan aminotransferase. Although the spontaneous breakdown of IPA has been considered (SHELDRAKE 1973), IP A decarboxylase activity was found in the supernatant fraction of tomato cell-free preparation (GIBsoN et al. 1972b). In this study, we estimated directly the IPA decarboxylase activity by assaying of carbon dioxide evolution, even though the amount of evoluted carbon dioxide was small. In contrast with IPA decarboxylase activity, the activity of pyruvic acid decarboxylase (EC 4.1.1.1.) in the extract of dark-grown epicotyl was much higher than that of light-grown one (unpublished). IAAld oxidase has been partially purified from Avena coleoptiles by RAJAGOPAL (1971). This enzyme is oxygen obligatory and PMS serves as hydrogen acceptor for aldehyde oxidation in the absence of molecular oxygen. On the other hand, WIGHTMAN and COHEN' (1968) reported the presence of an NADdependent IAAld dehydrogenase system in the cell-free preparation of mung bean seedlings. Pea IAAld oxidase is oxygen obligatory, but PMS and NAD did not stimulate the enzyme activity (unpublished). OUf experimental results suggested that the control of IAA biosynthesis from tryptophan under different light conditions is not likely to involve any very specific regulatory mechanism at the enzyme level in the first pathway. As shown in Table 3, the amine oxidase activity in pea seedlings grown in the light was one-fifth of those grown in the dark, suggesting that an increased IAA content in the dark-grown pea epicotyl is correlated with the high amine oxidase activity. However, the preparations of amine oxidase from pea seedlings catalyze the oxidation not only of tryptamine but also that of a variety of other amines, such as putrescine, spermidine and so on (HILL and MANN 1964). Therefore, it may closely related to the amount of tryptamine or its forming systems in pea seedlings, if amine oxidase functions as an enzyme in IAA biosynthesis according to the scheme shown in the second pathway. SCHNEIDER et al. (1972) reported that tryptamine is not detected in the shoot in both of tall and dwarf pea seedlings. Experiments investigating this problem are in progress in our laboratory. On the other hand, light-induced inhibition of amine oxidase activity in pea seedlings was also reported by MACHO LAN and MINAli(1974), although its mechanism is not elucidated. These results presented in the paper indicate that the increased IAA content in the dark conditions is not likely due to the activity of specific enzymes studied. However, it should be kept in mind that other enzymes involve in different pathways for IAA synthesis, except the above-mentioned enzymes might be closely related to the IAA production in the dark condition. Another possibility is that the amount of tryptophan or its related substances as the precursor of IAA in plant tissues differs significantly under light conditions.
42
Y. SUZUKI et al.
Acknowledgement This work has been partly supported by a Grant-in-Aid for Scientific Research (No. 244003) from the :M inistrv of Education, Science and Culture, .T apan.
References GIBSON, R. A., SCHNElDEH, E. A., and WIGHTMAN, F.: Biosynthesis and metabolism of illdole-3 acetic acid. II. In vivo experiments with HC-Iabelled precursors of IAA in tomato and barley shoots . .T. Exptl. Bot. 23, 381-399 (1972 a). BARRETT, G., and WIGHTl\L\K, F.: Biosynthesis and metabolism of indole-3-acetic acid. Ill. Partial purification and properties of a tryptamine-forming L-tryptophan decarboxylase from tomato shoots . .T. Exptl. Bot. 23, 775 -786 (1972 b). HILL, .T. M., and MANN, P . .T. G.: Further properties of the diamine oxidase of pea seedlings. Biochern. J. 91,171 - 182 (1964). HOLMSTEDT, B., LARSSON, L., and THAM, R.: Further studies of a spectrophotometric method for the determination of diamine oxidase activity. Biochim. Biophys. Acta 48, 182-186 (1961). KAMISAIL\, S., and LARSEN, P.: Improvement of the indolo-a-pyrone fluorescence method for quantitative determination of endogenous indole-3-acetic acid in lettuce seedlings. Plant & Cell Physiol. 18, 595 -602 (1977). LOWRY, O. H., ROSEBROlTGH, N. J., FARR, A. L., and RANDALL, R.: Protein measurement with the Folin-phenol reagent. J. BioI. Chem. 193, 265-275 (1951). MA CHOL ..\N, L., a,nd MINAR, J.: The depression of the synthesis of pea diamine oxidase due to light and the verification of its participation in growth processes using competitive inhibitors. Bio1. Phmt. (Praha) 18, 86-93 (1974). MASUDA, Y., KAMISAKA, S., YANAGISAWA, H., and SUZUKI, Y.: Effect of light on growth and metabolic activities in pea seedlings. Changes in cell wall polysaccharides during growth in the dark and light. Hiochem. Physiol. Pflanzen. 176, 23 -34 (1981). MATHERoN, M. E., and MOORE, T. C.: Properties of an aminotransferase of pea (Pisum sativum L.). Plant Physiol. 02, 63 -67 (1973). NISHIT.\1\I, K., and MASUDA, Y.: Growth and cell wall changes in azuki bean epicotyls I. Changes in wall polysaccharides during intact growth. Plant & Cell Physiol. 20, 63 -74 (1979). RAJAGOPAL, R.: Metabolism of indole-3-acetaldehyde. III. some characteristics of the aldehyde oxidase of Avena coleoptiles. Physiol. Plant. 24, 272-281 (1971). SACHS, J.: Uber den Einfluf3 der Lufttemperatur und des Tageslichtes auf die stiindlichen und taglichen Anderungen des Langewachstums (Streckung) der Internodien. Arb. Rotan. Inst. Wilrzburg 1, 99-192 (1872). SCHNEIDER, E. A., GIBSON, R. A., and WIGHTMAN, F.: Biosynthesis and metabolism of indole-3acetic acid. 1. The native indoles of barley and tomato shoots. J. Exptl. Bot. 23, 152 -170 (1972). SHELDRAKE, A. R.: The production of hormones in higher plants. BioI. Rev. 48, 509-559 (1973). SUZUKI, Y., and YANAGISA \VA, H.: Effect of neocuproine and cuprizone on the development of amine oxidase and growth in pea seedlings. Plant & Cell Physiol. 17, 1359 -1362 (197G). THIMANN, K. Y.: The natura.l plant hormones. In: "Plant Physiology", ed. STEWARD, F. C., vol. 6B "Physiology of Development: The Hormone." Academic Press, New York and London 1972. TRUELSEN, T. A.: Indole-3~pyruvjc acid as an intermediate in the conversion of tryptophan to indole3-acetie tteid. I. Some characteristi('s of tryptophan transaminase from mung bean seedlings. Physiol. Plant. 28, 289 -295 (1972).
IAA Content and IAA Biosynthesis Enzymes
43
- Indole-3-pyruvic acid as an intermediate in the conversion of tryptophan to indole-3-acetic acid. II. Distribution of tryptophan transaminase activity in plants. Physiol. Plant. 28, 67 -70 (1973). WWHnIANN, F., and COHEN, D.: Intermediary steps in the enzymatic conversion of tryptophan to IAA in cell free systems from mung bean seedlings. In: "Biochemistry and Physiology of Plant Growth Substances", ed. WIGHTMAN, F., and SETTERFIELD, G., The Runge Press, Ottawa, pp. 273-288b (1968).
Received May 27, 1980. Authors' address: Professor Dr. Y. SUZUKI, Department of Biology, Faculty of Science, Osaka City University, Sumiyoshi-ku, Osaka 558, Japan; Associate Professor Dr. S. KA}lISAKA, Ditto, Mr. H. YANAGISAWA, Ditto, Mr. S. MIYATA, Ditto, Professor Dr. Y. MASUDA, Ditto.
Effect of Light on Growth and Metabolic Activities in Pea Seedlings II. Changes in the IAA Content and Activities of Enzymes Involved in the IAA Biosynthesis during Growth Y. SUZUKI,
S.
KAMISAKA,
H.
YANAGISAWA,
S. MIYATA and
Y. MASUDA
Department of Biology, Faculty of Science, Osaka City University, Osaka, Japan Key Term Index: epicotyls, IAA, tryptophan aminotransferase, indolepyruvic acid decarboxylase, indoleaceta.ldehyde oxidase, amine (tryptamine) oxidase, light inhibition; P1:sum sativum.
Summary 1'he effect of light and dark conditions on the IAA content and the activities of tryptophan aminotransferase, indolepyruvic acid decarboxylase, indoleacetaldehyde oxidase and amine oxidase in pea (Pisum sativum L., cv. Alaska) seedlings was studied and the following results were obtained. 1. The endogenous IAA content was higher in shoots of dark-grown pea seedlings than in those of light-grown ones. When seedlings which had been grown in the dark were transferred to light, the endogenous IAA content decreased. When, on the other hand, the light-grown seedlings were transferred to darkness, the IAA content increased. 2. No significant difference in the effect of the light and dark conditions was found on the activities of tryptophan aminotransferase, indolepyruvic acid decarboxylase and indoleacetaldehyde oxidase in the epicotyl. 3. The activity in the apical part of amine oxidase which catalyzes the conversion of tryptamine to indoleacetaldehyde was inhibited considerably under light conditions. These results indieate that the increased IAA content in the dark conditions is not likely to depend on specific regulatory mechanisms at the enzyme level. Although the amine oxidase activity was found to be high in the dark-grown pea seedlings, the function of this enzyme in IAA biosynthesis remains unsolved.
Introduction
Growth and development in higher plants are under the genetic control and also under the environmental control, the former being often regulated by the latter, such as light conditions (cf. SACHS 1872). In our previous paper (MASUDA et al. 1981) we reported that the mechanical property of the epidermal cell wall and the extension potential of the inner tissue of the internodes are remarkably influenced by light conditions. We also found that the metabolic turnover of sugars of cell wall polysaccharides, particularly of neutral sugars in the pectic fraction, is closely related with the extension of the internodes as affected by light conditions (NISHITANI and MASUDA 1979). Plant growth substances play roles as effectors in the regulation of growth and development. Among the substances, auxin is believed to playa leading role in the reguAbbreviations: IAA, indole-8-acetic acid; EDTA, sodium ethylenediamine tetraacetate; IAAld, indoleacetaldehyde; IPA, B-indolepyruvic acid; TPP, thiamine pyrophosphate; TCA, trichoroacetic acid; PMS. phenazine mcthosulphate.
36
Y.
SUZUKI
et al.
lation (THIMANN 1972). However, the correlation of intact growth in plants with auxin metabolism, synthesis and degradation under different environmental conditions has not been well established. Thus, we decided to investigate changes in the IAA content and in the activities of different enzymes involved in the IAA biosynthesis in pea seedlings grown in darkness and in light. This study tries to correlate shoot growth under different light conditions with auxin metabolism. Material and Methods Plant material Pea (Pisum sativum L., cv. Alaska) seeds were sown and germinated in moist vermiculite in plastic trays at 25 00. The trays were kept under continuous light (ca. 3,000 lux at plant level) or in total darkness. The seedlings in another tray were first grown in the dark for 5 days and then transferred to light and kept for 2 days, or they were first grown in the light then transferred to darkness.
Preparation of enzyme frtwtions 1. Tryptophlm aml~notransftrase: The exeised shoots, surface sterilized for 10 min in 1 % benzalkonium chloride solution and then washed thoroughly with deionized water, were homogenized in a mortar with three volumes (vjw) of cold 0.1 M phosphate buffer (pH 7) containing 10 mM 2-mercaptoethanol, 1 mM EDTA, 10 l,M pyridoxal phosphate and wet Polyclar AT (shoots: Polyclar AT = 1: 0.1 in w/w). The resulting slurry was passed through a layer of cotton cloth and then centrifuged for 20 min at 10,000' g. The supernatant was fraetionated with ammonium sulfate, and the precipitate obtained with 80 % saturation was dissolved in a minimal amount of 50 roM phosphate buffer (pH 8) containing 10 mM 2-mercaptoethanol, 1 mM EDT A and lO.uM pyridoxal phosphate. After centrifugation for 30 min at 10,000 . g, the supernatant was passed through a column (2.2 x 65 cm) of Sephadex G-25, then the protein solntion eluted was used for the enzyme assay. 2. IP.!! decarboxylase: The excised shoots were homogenized in a Waring blender with twice volumes (v/w) of 0.1 M phosphate buffer (pH 7) containing 1 roM cysteine HOI, 0.1 roM EDTA, 0.1 mM TPP, 1 mM MgS0 4, 20% (vjv) glycerol and PolycIar AT (shoots: Polyclar AT = 1: 0.06 in wjw). The homogenate was filtered through a layer of cotton cloth and centrifuged for 30 min at 10,000· g. The superna.tant was fractionated with ammonium sulfate and the precipitate obtained at 80 % saturation was dissolved in a minimum amount of 50 mM phosphate buffer (pH 7) containing 10- :>.M TPP and 1 mM MgS04 and then dialyzed overnight against 1,000 ml of the same buffer solution with three changes. The dialysate was used for the enzyme assay. 3. I AAld oxidase: The enzyme was prepared from the excised shoots and the procedure was the same as those of IPA decarboxylase preparation, except that TPP and MgS04 were not included in the extraction medium. 4. Amine o:cidase: The excised shoots were homogenized in a mortar with twice volumes (v/w) of cold 0.1 M phosphate buffer (pH 7). The homogenate was filtered through a layer of cotton cloth a,nd centrifuged for 20 min at 10,000· g. The supernatant was used for the enzyme assay.
Determination of endogenous IAA. IAA in pea shoots was extracted with methanol. The purification of IAA in the methanol extract was carried out as deseribed earlier (KAMISAKA and LARSEN 1977). 1-14C-IAA (3,000 dpm) was added to the methanol extract to correct IAA loss during purification, then the methanol fraction was evaporated under reduced pressure at 40 °0. To the residue, 10 ml of 0.5 M K2 HPO, solution was added. Lipoidal substances were extracted three times with light petrolium ether and twic.e with ethyl ether. Then, the pH of the solution was adjusted to 3 with 2.8 M Hg P04 solution. The acidic fraction was shaken with ethyl ether. The ethyl ether fraction was a.gain shaken with 10 ml
IAA Content and IAA Biosynthesis Enzymes
37
of 0.05 M KzHP0 4 solution. After adjusting the pH of the 0.05 M KzHP04 fraction to 3 with 0.28 M H3 P0 4 solution, the acidic ethyl ether fraction was prepared by sha,king the KzHP0 4 fraction with ethyl ether. The determination of IAA in the acidic ether fraction was carried out by the modified indolo-{X-pyrone fluorescence method of KAMISAKA and LARSEN (1977).
Enzyme assays 1. Tryptophan aminotransferase: For the estimation of tryptophan aminotransferase activity, indolepyruvate-borate complex, the product of the enzyme action in the presence of borax-HCI buffer was assayed spectrophotometrically according to the method of TRUELSEN (1972) and that of MATHERON and MOORE (1973). The routine reaction mixtures consisted of 0.5 ml of enzyme preparation, 1 ml of 0.5 M borax-HCl buffer solution (pH 8.5), 0.4 ml of 0.1 M tryptophan (in 0.2 N HCI), 0.1 ml of 0.2 M {X-ketoglutarate (pH 6.5) and 0.1 ml of 1 mM pyridoxal phosphate solution. Total volume was adjusted to 2.5 ml with deionized water. After reaction mixtures were incubated for 60 min at 30 °c with shaking, the reaction was stopped by the addition of 0.5 ml of 24 % TCA. The mixtures were then centrifuged for 10 min at 3,000 rpm. Two ml of each supernatant was added to 2 ml of 1 M sodium arsenate-boric acid mixture (pH 6.5) separately and allowed to stand for 20 min. Then the absorbance was measured at 328 nm. Reaction mixtures minus {X-ketoglutarate were used as controls. 2. J P A decarboxylase activity: For the estimation of IP A decarboxylase activity, the evolution of carbon dioxide caused by IPA in the presence of enzyme was assayed manometrically at 30°C. The reaction mixture consisted of 2 ml of enzyme solution, 0.3 ml of 10 mM TPP and 10 mM IP A (adjusted to pH 7 with 1 M phosphate buffer) in the main chamber. Side-arm contained 0.2 ml of 2 N H 2 SOt which was transferred to the main chamber at the end of the experiment. Control was used without the addition of IPA. The center well contained 0.2 ml of 15 % KOH or 2 N HzSO.1• The data were calculated from the carbon dioxide evolution after 2 h. 3. J A..4~d oxidase: The enzyme activity was estimated by determining the oxygen upta.ke from air in a Wa,rburg flask at 30°C. The reaction mixture was composed of 1 ml of 0.1 M phosphate buffer (pH 7), 1 ml of enzyme solution and 0.3 ml of 20 mM IAAld bisulphite (side-arm). Control was used without the addition of substrate. The center well contained 0.1 ml of 15% KOH. The data were cahmlated from the oxygen uptake in the first 20 min. 4. Aml:ne oX1:dase: Amine oxida.se activity for tryptamine and putrescine was estimated using a. Clark oxygen electrode at 30°C and the consumption of soluble oxygen was measured with a. TOA polyrecorder (Model 10 A) (SUZUKI and YANAGISAWA 1976). The standard assay system was as follows: 0.4 ml of enzyme preparation, 1 mlof 0.1 M phosphate buffer (pH 8), 0.1 ml of beef liver crystalline catalase (250 p.gjO.l ml) and 0.5 ml of tryptamine· HCl or putrescine· 2 HCl (50 mM each). Total volume was adjusted to 3.2 ml with deionized water. Colorimetric estimation of the enzyme activity for putreseine was also assayed by a method similar to that described by HOLMSTED'f et aJ. (1961). The reaction mixture consisted of 0.5 ml of 50 mM putrescine· 2 HCl solution, 0.1 ml of 0.1 M phosphate buffer (pH 7), 0.2 m] of enzyme preparation, 0.2 rul of 0.1 % o-aminobenzaldehyde solution (in ethanol) and 0.1 m] crysta11ine beef liver catalase (250 !lg/0.1 ml). Total volume was adjusted to 2.6 ml with deionized water. The mixture was incubated for 5 min at 30°C with shaking, and the reaction was stopped by adding 1 ml of 50 % TCA, then 4 ml of deionized water was added. The mixture was filtered off after standing for 30 min in an ice box, then the absorbance of the filtrate was estimated at 435 nm. Both reactions were started by the addition of enzyme preparations.
Protein determination The protein content was determined by the method of LOWRY et a1. (1951) using bovine serum albumin as the standard.
38
Y.
SUZUl\l
et al.
Results
Effect of dark and light on the IAA content in pea seedlings The effects of different regimes of the dark and light on the IAA content in pea shoots were determined as shown in Table 1. When the dark-grown pea seedlings were exposed to light, the IAA content per fresh weight or per shoot usually was lower than that of the seedlings grown in the dark for 5 + 2 days. However, if the reverse order of the light treatment was used, the IAA content increased in the dark, suggesting that the dark condition stimulated the synthesis of IAA or prevented its decomposition. In contrast, light might induce the conditions of IAA destruction or inhibition of IAA synthesis. Table 1. The endogenous I AA content in Alaska pea shoots grown in the dark and light. Pea seedlings were grown ill the dark and light and shoots were subjected to the IAA determinatioH. D5: grown for 5 days in the dark, D2: grown for 2 days in the dark, L2: grown for 2 days in the light L8: grown for 8 days in the light. Experiment
IAA content
Growth conditions
ng/g fresh weight
I
II
8.4 ± 0.6 14.6 ± 0.7 10.9 ± 1.8 32.3 ± 2.1 42.5 ± 1.5
D5 D5 ~ D2 D5 ~ L2 18 L8 ~ D2 L8 -,.. 1.2
30.9
±
2.0
ng/shoot
1.7
±
0.1 5.1 ± 0.2 2.6 ± 0.4 6.5 ± 0.4
11.9 8.1
± 0.4
-+:
0.5
Activities of tryptophan aminotransferase, IP A decarboxylase and IAAld oxidase in the dark- and light-grown pea seedlings Extracts from apical parts of dark- and light-grown pea seedlings were examined for the activity of tryptophan aminotransferase, IP A decarboxylase and IAAld oxidase. As shown in Table 2, no significant difference in the both conditions of light and dark was found for the activity of these enzymes in the extract of apical segments. Table 2. Activities of tryptophan aminotransferase, IP A decarboxylase and IAAld oxidase in Alaska
pea epicotyls grown in the dark and light. Pea seedlings were grown in the dark and light for 5 to 6 days or 7 to 8 days, and epicotyls were subjected to enzyme activity determination. Epicotyl length em
Growth conditions
7.4 ± 0.6 19.8 ± 2.5 7.2 ± 0.5
L7-8 D7-8 D5-6
Enzyme activities Tryptophan aminotransfera,se IPA nmoles/hr(mg protein
IP A decarboxylase IAAld oxidase ,ul CO 2(hrjmg ,Ill 02/hr/mg protein protein
10
0.16 0.11
13 15
4.56
5.10
39
IAA Content and IAA Biosynthesis Enzymes
Effect of light on amine oxidase (tryptamine oxidase) activity in the dark-grown pea seedlings Contrary to the above enzymes, the activity of tryptamine oxidase in the darkgrown pea epicotyls was five-fold as compared with that in light-grown ones, if the comparison was made on the same length of the epicotyl (Table 3). When the seedlings which had been grown for 5 days in the dark were transferred to light and kept for 2 days, the amine oxidase activity in the epicotyl was significantly decreased. However, such trend was not observed for tryptophan aminotransferase activity (Table 4). Table 3. Activity of amine oxidase in Alaska pea seedlings grown in the dark and light. Pea seedlings were grown for [) or 8 days in the dark and light and then epicot.yls were subjected to the enzyme activity determination. Epicotyl length (cm)
7.2
± 0.5
7.4. ± 0.6 2.9 :i-: 0.5
Growth ronditions
D5 L8 L5
Sped fie activity (nmole 02/min!mg protein) Tryptamine
Putrescine
21 4
188 22
12
41
Table 4. Activities of amine oxidase and tryptophan aminotransferase in Alaska pea seedlings grown
in the dark and light. Pea seedlings were grown for 5 or fnrther for 2 days in the dark and light and then epicotyls were subjected to enzyme activity determinations. Specific activities Epicotyl length cm
7.2 ± 0.5 19.8 ± 2.5 10.6 ± 1.7 2.9 ± 0.5 7.4±O.6 9.8 ± 0.8
Growth ('onditions
Amine oxidase (nmole 0z/ minjmg protein)
Tryptophan aminotra,nsferase (IPA nmolesjhrjmg
Tryptamine
Putrescine
protein)
D5
21
188
])5 --+ D2
23
252
I
113
12
41
:J
9'> '"'
15.3 13.3 13.5 10.1 10.0 13.0
Db --+ L2 15 I~5 --+ 13 15 -~ D2
,..,
U
-
67
Effect of dark and light on the distribution of amine oxidase activity in different shoot segments of pea seedlings In order to examine the distribution of amine oxidase activity in different shoot segments, different parts of the pea seedlings grown in the dark and light were excised
40
Y.
SUZUKI
et al.
Table 5. Activity of amine oxidase in Alaska pea seedl1:ngs grown in thp. dark and liyht. Pea seedlings were grown for 5 or 8 days in the dark and light a,nd ep:ieotyls were subjected to the enzyme activity determinations. A: apical part, M: middle part, B: basal part Epicotyl length em
7.2
±
0.5
10.6± 1.7 7.4 ± 0.6
Growth conditions
D5 D5 .- L2 L8
Enzyme a.ctivity Tryptamine (nmoles 02/minjmg protein)
Putrescine (nmoles Ll-pyrroline/ min/mg protein)
A
M
B
A
M
B
25 23 13
99 -~
19
19 4
6
425 425 285
285 301 65
81 48
4
27
and then separately analyzed. As shown in Table 5, the highest enzyme activity was found in the apical parts of the internode in the both conditions of dark and light. When the seedlings which had been grown in the dark for 5 days were transferred to the light, the ability of tryptamine oxidation in the basal part of the internode was significantly depressed as compared with the apical and middle parts of the internodes. In our experiments, the depression of amine oxidase activity was fairly large in the case of tryptamine oxidation rather than that of putrescine when the plant grown in the dark was exposed to the light (Table 5).
Discussion
We have found in the present study that there is a good correlation between the IAA content in the extract of epicotyl (Table 1) and epicotyl elongation (Table 2). The amount of 1AA in the dark-grown epicotyl as compared with that of light-grown one is relatively high. The reversible inhibition of the internode elongation of pea seedlings by light was also well correlated with the changes in the 1AA content (Table 1). Therefore, it appears that elongation of the epicotyl of pea seedlings is controlled by light via the control of 1AA content. On the other hand, the biosynthesis of 1AA from tryptophan has been considered to be carried out mainly via two different pathways, i.e. tryptophan -* indolepyruvic acid -* indoleacetaldehyde -* 1AA (first pathway) and tryptophan -* tryptamine -+ indoleacetaldehyde -+ 1AA (second pathway) (SHELDRAKE 1973). The pathways of 1AA biosynthesis in mung bean, barley and tomato have been studied extensively by Wightman and his co-workers (SCHN'EIDER et al. 1972; GIBSON et al. 1972a, b). They partially purified tryptophan aminotransferase and tryptophan decarboxylase from tomato shoots. However, the correlation of intact growth with auxin metabolism, synthesis and degradation under different environmental conditions such as light and darkness has not been studied. If the 1AA content depends on the rate of 1AA biosynthesis, we might expect that some enzyme activities related
IAA Content and IAA Biosynthesis Enzymes
41
to IAA biosynthesis in dark-grown pea seedlings differ from those grown in the light. As shown in Table 2, no significant difference in the both conditions of light and dark was found for the activity of three key enzymes involved in the first pathway of IAA biosynthesis, namely tryptophan aminotransferase, IPA decarboxylase and IAAld oxidase. TRUELSEN (1973) has shown that tryptophan aminotransferase activity in pea shoot tips which were grown in the light is about two-fold as compared with those grown in the dark. We have also found that in the pea seedlings light tends to increase the activity of tryptophan aminotransferase. Although the spontaneous breakdown of IPA has been considered (SHELDRAKE 1973), IP A decarboxylase activity was found in the supernatant fraction of tomato cell-free preparation (GIBsoN et al. 1972b). In this study, we estimated directly the IPA decarboxylase activity by assaying of carbon dioxide evolution, even though the amount of evoluted carbon dioxide was small. In contrast with IPA decarboxylase activity, the activity of pyruvic acid decarboxylase (EC 4.1.1.1.) in the extract of dark-grown epicotyl was much higher than that of light-grown one (unpublished). IAAld oxidase has been partially purified from Avena coleoptiles by RAJAGOPAL (1971). This enzyme is oxygen obligatory and PMS serves as hydrogen acceptor for aldehyde oxidation in the absence of molecular oxygen. On the other hand, WIGHTMAN and COHEN' (1968) reported the presence of an NADdependent IAAld dehydrogenase system in the cell-free preparation of mung bean seedlings. Pea IAAld oxidase is oxygen obligatory, but PMS and NAD did not stimulate the enzyme activity (unpublished). OUf experimental results suggested that the control of IAA biosynthesis from tryptophan under different light conditions is not likely to involve any very specific regulatory mechanism at the enzyme level in the first pathway. As shown in Table 3, the amine oxidase activity in pea seedlings grown in the light was one-fifth of those grown in the dark, suggesting that an increased IAA content in the dark-grown pea epicotyl is correlated with the high amine oxidase activity. However, the preparations of amine oxidase from pea seedlings catalyze the oxidation not only of tryptamine but also that of a variety of other amines, such as putrescine, spermidine and so on (HILL and MANN 1964). Therefore, it may closely related to the amount of tryptamine or its forming systems in pea seedlings, if amine oxidase functions as an enzyme in IAA biosynthesis according to the scheme shown in the second pathway. SCHNEIDER et al. (1972) reported that tryptamine is not detected in the shoot in both of tall and dwarf pea seedlings. Experiments investigating this problem are in progress in our laboratory. On the other hand, light-induced inhibition of amine oxidase activity in pea seedlings was also reported by MACHO LAN and MINAli(1974), although its mechanism is not elucidated. These results presented in the paper indicate that the increased IAA content in the dark conditions is not likely due to the activity of specific enzymes studied. However, it should be kept in mind that other enzymes involve in different pathways for IAA synthesis, except the above-mentioned enzymes might be closely related to the IAA production in the dark condition. Another possibility is that the amount of tryptophan or its related substances as the precursor of IAA in plant tissues differs significantly under light conditions.
42
Y. SUZUKI et al.
Acknowledgement This work has been partly supported by a Grant-in-Aid for Scientific Research (No. 244003) from the :M inistrv of Education, Science and Culture, .T apan.
References GIBSON, R. A., SCHNElDEH, E. A., and WIGHTMAN, F.: Biosynthesis and metabolism of illdole-3 acetic acid. II. In vivo experiments with HC-Iabelled precursors of IAA in tomato and barley shoots . .T. Exptl. Bot. 23, 381-399 (1972 a). BARRETT, G., and WIGHTl\L\K, F.: Biosynthesis and metabolism of indole-3-acetic acid. Ill. Partial purification and properties of a tryptamine-forming L-tryptophan decarboxylase from tomato shoots . .T. Exptl. Bot. 23, 775 -786 (1972 b). HILL, .T. M., and MANN, P . .T. G.: Further properties of the diamine oxidase of pea seedlings. Biochern. J. 91,171 - 182 (1964). HOLMSTEDT, B., LARSSON, L., and THAM, R.: Further studies of a spectrophotometric method for the determination of diamine oxidase activity. Biochim. Biophys. Acta 48, 182-186 (1961). KAMISAIL\, S., and LARSEN, P.: Improvement of the indolo-a-pyrone fluorescence method for quantitative determination of endogenous indole-3-acetic acid in lettuce seedlings. Plant & Cell Physiol. 18, 595 -602 (1977). LOWRY, O. H., ROSEBROlTGH, N. J., FARR, A. L., and RANDALL, R.: Protein measurement with the Folin-phenol reagent. J. BioI. Chem. 193, 265-275 (1951). MA CHOL ..\N, L., a,nd MINAR, J.: The depression of the synthesis of pea diamine oxidase due to light and the verification of its participation in growth processes using competitive inhibitors. Bio1. Phmt. (Praha) 18, 86-93 (1974). MASUDA, Y., KAMISAKA, S., YANAGISAWA, H., and SUZUKI, Y.: Effect of light on growth and metabolic activities in pea seedlings. Changes in cell wall polysaccharides during growth in the dark and light. Hiochem. Physiol. Pflanzen. 176, 23 -34 (1981). MATHERoN, M. E., and MOORE, T. C.: Properties of an aminotransferase of pea (Pisum sativum L.). Plant Physiol. 02, 63 -67 (1973). NISHIT.\1\I, K., and MASUDA, Y.: Growth and cell wall changes in azuki bean epicotyls I. Changes in wall polysaccharides during intact growth. Plant & Cell Physiol. 20, 63 -74 (1979). RAJAGOPAL, R.: Metabolism of indole-3-acetaldehyde. III. some characteristics of the aldehyde oxidase of Avena coleoptiles. Physiol. Plant. 24, 272-281 (1971). SACHS, J.: Uber den Einfluf3 der Lufttemperatur und des Tageslichtes auf die stiindlichen und taglichen Anderungen des Langewachstums (Streckung) der Internodien. Arb. Rotan. Inst. Wilrzburg 1, 99-192 (1872). SCHNEIDER, E. A., GIBSON, R. A., and WIGHTMAN, F.: Biosynthesis and metabolism of indole-3acetic acid. 1. The native indoles of barley and tomato shoots. J. Exptl. Bot. 23, 152 -170 (1972). SHELDRAKE, A. R.: The production of hormones in higher plants. BioI. Rev. 48, 509-559 (1973). SUZUKI, Y., and YANAGISA \VA, H.: Effect of neocuproine and cuprizone on the development of amine oxidase and growth in pea seedlings. Plant & Cell Physiol. 17, 1359 -1362 (197G). THIMANN, K. Y.: The natura.l plant hormones. In: "Plant Physiology", ed. STEWARD, F. C., vol. 6B "Physiology of Development: The Hormone." Academic Press, New York and London 1972. TRUELSEN, T. A.: Indole-3~pyruvjc acid as an intermediate in the conversion of tryptophan to indole3-acetie tteid. I. Some characteristi('s of tryptophan transaminase from mung bean seedlings. Physiol. Plant. 28, 289 -295 (1972).
IAA Content and IAA Biosynthesis Enzymes
43
- Indole-3-pyruvic acid as an intermediate in the conversion of tryptophan to indole-3-acetic acid. II. Distribution of tryptophan transaminase activity in plants. Physiol. Plant. 28, 67 -70 (1973). WWHnIANN, F., and COHEN, D.: Intermediary steps in the enzymatic conversion of tryptophan to IAA in cell free systems from mung bean seedlings. In: "Biochemistry and Physiology of Plant Growth Substances", ed. WIGHTMAN, F., and SETTERFIELD, G., The Runge Press, Ottawa, pp. 273-288b (1968).
Received May 27, 1980. Authors' address: Professor Dr. Y. SUZUKI, Department of Biology, Faculty of Science, Osaka City University, Sumiyoshi-ku, Osaka 558, Japan; Associate Professor Dr. S. KA}lISAKA, Ditto, Mr. H. YANAGISAWA, Ditto, Mr. S. MIYATA, Ditto, Professor Dr. Y. MASUDA, Ditto.