Inhibitory Effects of Monoamine Oxidase Inhibitors on the Growth of Pea and Rice Seedlings

Plant & Cell Physiol. 12: 551-558 (1971)

Inhibitory effects of monoamine oxidase inhibitors on the growth of pea and rice seedlings

MIEKO KIMURA and CHIKAKO TANAKA Downloaded from https://academic.oup.com/pcp/article/12/4/551/1886246 by guest on 28 September 2021

Department of Pharmacology, School of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan

(Received May 6, 1970)

Two monoamine oxidase inhibitors of the hydrazine-type, safrazine and nialamide, inhibited growth in seedlings of rice and pea. We demonstrated histochemically that monoamine oxidase is located chiefly in sieve tubes and in the epidermis of pea seedling. Activity of this enzyme was high in the apical part of the epicotyl, decreasing toward the base. Inhibition of pea monoamine oxidase by safrazine and nialamide was observed histochemically and with an extract from the epicotyl. This supports the hypotfiesis that indole-3-acetic acid (IAA) is formed from tryptamine by amine oxidase and that inhibition of this enzyme causes lowering of the auxin level, resulting in growth inhibition. Inhibition of growth in rice seedlings by safrazine was reversed by the addition of IAA to the culture medium. A growth retardant, iV-dimethylamino succinamic acid (B995) differs in its chemical structure from other growth retardants, i.e. (2-chloroethyl)-trimethyl- ammonium chloride (CCC), 2,4-dichlorobenzyltributylphosphonium chloride (Phosfon) and 4-hydroxy-5-isopropyl-2-methylphenylammonium chloride, 1-piper- idine carboxylate (Amo-1618). The chemical structure of B995 is rather close to that of the hydrazine-type monoamine oxidase inhibitors, such as /9-piperonyl- isopropyl hydrazine (safrazine), iV-isonicotinoyl-iVy-[/8-(iV'-benzylcarboxyamido) ethyl]-hydrazine (nialamide). Monoamine oxidase inhibitors were demonstrated in animal tissues (1). Reed (2) reported that B995 inhibited the activity of amine oxidase in pea seedling. Hence, the application of monoamine oxidase inhibitors to growing plants may be expected to result in reduction of amine oxidase activity with a subsequent decrease in IAA production, leading to growth retardation. The growth inhibition, thus, caused would be overcome by giving an adequate amount of IAA. The purpose of the present investigation was to demonstrate the localization of amine oxidase in pea seedlings using histochemical technique, and to find a relation between this enzyme and auxin production.

Metarials and methods

Plant materials After sterilization of pea seeds (Pisum sativum L. cv. Alaska) and rice seeds (var. Kyoto Asahi) with 75% ethanol for 15 min, the samples were soaked in water 551 552 M. KIMURA and C. TANAKA for 12 hr. Seeds were then incubated in a medium containing growth retardants or monoamine oxidase inhibitors for 24 hr at room temperature. Then pea seeds were transplanted in sand and allowed to grow in the dark at 25-27°C. Rice seeds were planted in water and allowed to grow at 25-27°C. In each experiment 10 seedlings were used.

Amine oxidase assays Downloaded from https://academic.oup.com/pcp/article/12/4/551/1886246 by guest on 28 September 2021 Seedlings of peas, grown for 12 days, were used for histochemical experiments. Seedlings were put in a test tube and frozen with a mixture of acetone and dry ice. Transverse sections, 10 to 20 p thick, were excised from the upper region of each of the first, second and third internodes, in a cryostat at —17°C. Sections were dried for several minutes at room temperature. Amine oxidase was demonstrated using the method of Glenner et al. (3). The incubation medium consisted of 25 mg tryptamine hydrochloride, 4 ml of 0.1 % nitroneotetrazolium chloride, 4 ml of 0.1 % NazSO*, 5 ml 0.1 M phosphate buffer (pH 8.1) and 1 ml distilled water. Growth retardants (CCC and B995), as well as safrazine and nialamide, were added to the incubation medium at a concentration of 5 x 10~3M. NO substrate was added for the control incubation medium. Sections were incubated for 4 hr at 25°C, then were washed with 0.1 M phosphate buffer. These were fixed in 10% neutral formalin for 1 to 24 hr, and mounted in buffered glycerin (phosphate buffer 1: glycerin 9). One gram of epicotyl excised from 7-day old pea seedlings was homogenized in 25 ml of borate-phosphate buffer (0.033 M, pH 8.1) (4) in a glass homogenizer. This homogenate was centrifuged at 1000 X^ for 15 min. Amine oxidase activity in the supernatant was measured by the modified method (2) of Wurtmann and Axelrod (5). Tryptamine-2-uC bisuccinate (specific activity 8.9 mCi/mmole, New England Nuclear Corp.) was used as substrate. The following assay conditions were used (5). The incubation mixture consisted of tryptamine-2-"C bisuccinate 0.04m^mole (9776 cpm) in 0.1 ml borate-phosphate buffer (0.033 M, pH 8.1), an inhibitor solution in 0.1 ml borate buffer, 0.1 ml enzyme preparation (containing 2.6mg/ml protein), and borate-phosphate buffer giving a final volume of 0.4 ml. The mixture was incubated for 30 min at 30°C with continuous shaking, then the reaction was stopped by the addition of 0.2 ml of 2 N HC1. The indoleacetoaldehyde formed was extracted with 6 ml toluene by shaking the mixture for 5 min. After centrifugation, 4 ml of the organic layer was transferred to a vial containing 10 ml Bray's solution, and its radioactivity was counted for 2 min with a Beckman liquid scintillation spectrophotometer.

Results Safrazine inhibited germination and seedling growth in peas (Table 1). 10"2 M B995 also markedly suppressed germination of peas but 10~2 M CCC did not. Shoot growth in rice seedlings was inhibited by safrazine and nialamide; the inhibitory effect of the former being somewhat stronger than that of the latter (Fig. 1 A and B). Compared with the control, the shoot was more stiff and leaves were deeper green in color. Root growth in rice seedlings was somewhat promoted by these compounds at certain concentrations (Fig. 1A and B). The number of roots also increased. Time courses of shoot and root growth are shown in Table 2. Inhibitory effects of monoaraine oxidase inhibitors on the growth of pea and rice seedlings 553 rh

rh 1 i Downloaded from https://academic.oup.com/pcp/article/12/4/551/1886246 by guest on 28 September 2021 •h

Fig. 1. Effects of safrazine {A) and nialamide (B) on the growth of rice seedlings measured after 8 days of water culture.

cent. Iff^O"5 O* K)'3 »2 M ccrtO"6 ti5 O* B3 tf2 shoot r root concentration of drugs

Table 1 Inhibitory effects of 24 hr seed treatment with safrazine on germination and seedling growth in peas Safrazine cone Shoot length " Germination (M) (cm) (%) 0 3.95 + 0.42(100.0) 100 io-6 3.68+0.46 ( 93.4) 100 io-5 2.86 + 0.45( 72.4) 100 10-4 2.37 + 0.37 ( 60.0) 100 io-3 1.48+0.21 ( 37.5) 100 io-2 0 0 " Measured after 7 days of sand culture following safrazine treatment. Average of 10 seedlings with S. E. M. and % of control in parentheses.

Table 2 Effects of safrazine on the growth of shoot and root in rice seedlings 0 a Safrazine Shoot length (cm) Root length (cm) cone 7 12 15 7 12 15 (M) (days) (days) 0 3.04+0.07 7.83+0.49 11.60±0.46 4.68±0. 17 8.87+0.59 10.41 ±1.07 10"6 3.29 + 0.32 7.95 + 0.47 12.57+0.49 3.63 + 0.33 8.33 ±0.56 10.59±0.79 io-5 2.50±0.22 7.09 + 0.48 12.24±0.41 5.17±0.43 7.75±0.63 9.20±0.67 10"4 1.95+0.13 8.05+0.36 11.28±0.70 4.74±0.43 9.14 + 0.77 9.84+0.83 10"3 1.60 + 0.11 4.21+0.34 7.46±0.24 4.44±0.29 6.55+0.61 8.34±0.60 io-2 1.30 + 0.11 3.13+0.09 4.76±0.19 2.13 + 0.25 4.04±0.34 6.60±0.58 " Average of 10 seedlings ±S. E. M. 554 M. KIMURA and C. TANAKA

Shoot growth and root growth in rice seedlings was very weakly inhibited by B995 (Table 3). Histochemical studies were performed using sections excised from the first, second and third internodes. Formazan glanules, considered to result from amine oxidase activity, were abundant in the epidermis and sieve tubes of the third internode (Plate 1) and in the cotyledon. Amine oxidase activity was lower in the second Downloaded from https://academic.oup.com/pcp/article/12/4/551/1886246 by guest on 28 September 2021

Plate 1. Amine oxidase activity in the third internode of pea seedling. Upper: substrate added ( X 40). High activity is seen in epidermal cells and sieve tubes. Lower: no substrate added (x 40). Inhibitory effects of monoamine oxidase inhibitors on the growth of pea and rice seedlings 555

Table 3 Effects of B 995 on the growth of shoot and root in rice seedlings a B995 Shoot length (cm)" Root length (cm) cone 7 12 15 7 12 15 (M) (days) (days) 0 3.04±0.07 7.83+0.49 11.60±0.46 4.68±0. 17 8.87 + 0.59 10.41 + 1.07 10-6 2.94±0. 14 8. 31±0.49 10. 77±0.85 5.27±0.26 8.55+0.62 11.34 + 0.87 io-5 2.63±0.14 8.47±0.67 12.30 + 0.47 4. 77±0.23 9.15±0.63 9.82 + 0.94 Downloaded from https://academic.oup.com/pcp/article/12/4/551/1886246 by guest on 28 September 2021 io-« 2.39±0. 13 8.55±0.39 11.03 + 0.95 4. 53±0.47 8. 76±0. 78 9.75+0.48 10"3 2.30±O. 12 7.98±0.49 11.05 + 0.71 4.37 + 0.24 7.00+0.33 7.87+1.68 lO"2 2.12±0.16 7.48±0.29 10.87±0.32 3.43±0.26 7. 18±0. 71 7.90+0.44 Average of 10 seedlings ± S. E. M.

shoot cent. 8

cont. 1CT3M tT!M cont 10"3M KJ"2M concentration of safrazine(mol/I) Fig. 2 Effects of safrazine and IAA on the growth of rice seedlings

. .,.; / TV - K7 \ Plate 2. ^4min« oxidase activity m the second mternode oj pea seedling (X40). §56 M. KIMURA and C. TANAKA

Table 4 Effects of growth retardants on amine oxidase activity in pea seedling Inhibitors (5X10"3M) Grade of staining None ccc B995

Safrazine # Downloaded from https://academic.oup.com/pcp/article/12/4/551/1886246 by guest on 28 September 2021 Nialamide No substrate

internode (Plate 2), and was very weak in the first internode; though some activity- was demonstrated in sieve tubes (Plate 3). At 5 x 10~3 M, nialamide and safrazine inhibited amine oxidase severely, B995 moderately and CCC not at all (Table 4). Safrazine almost completely inhibited indoleacetaldehyde formation by the extract from pea seedlings at concentrations of 10~2, 10"3, 10"4 and 10"5 M. How- ever, it slightly stimulated activity at 10~7 and 10~a M (Table 5). The inhibitory effect of nialamide on amine oxidase activity was weaker than that of safrazine (Table 6). After sterilizing and soaking them in water for 12 hr, rice seeds were incubated in 10"2 and 10"3 M safrazine solutions for 24 hr, then were cultured in 10~5 and 10~6 M IAA solution. Suppression of shoot growth by safrazine was reversed by IAA, as shown in Fig. 4. Elongation of roots was occasionally promoted by safrazine, but was rather inhibited by IAA.

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Plate 3. Amine oxidase activity in the first intemode of pea seedling (x40). Inhibitory effects of monoamine oxidase inhibitors on the growth of pea and rice seedlings 557

Table 5 Inhibitor): effect of sa/razine on indoleacetaldehyde formation ' from tryptamine-2-ltC by pea seedling extract Cone of Radioactivity Indoleacetaldehyde Inhibition safrazine of extract formed (M) (cpm) (mjumole) 0 5170 + 102 0.02114 0 Downloaded from https://academic.oup.com/pcp/article/12/4/551/1886246 by guest on 28 September 2021 io-s 7253 + 717 0.02967 -29 10-' 6644 ±958 0.02715 -23 10"6 1043± 42 0.00426 80 10"5 12± 7 0.00044 98 io-* 2.3±3.5 0.00009 99-100 10-3 0.5±3.0 0.000019 99-100 io-2 0.2+4.3 0.000008 99-100 0.4x10"' mole tryptamine-2-14C was contained in the reaction mixture. Indoleacetaldehyde formed was calculated by correcting observed cpm values for the blank value (98 cpm) obtained by incubating tryptamine- 2-14C with enzyme solution boiled for 10 min. Each value is the mean of three samples.

Table 6 Inhibitory effect of nialamide on indoleacetaldehyde formation' from tryptamine-2-uC by pea seedling extract Cone of Radioactivity Indoleacetaldehyde nialamide of extract formed ,0, •. (M) (cpm) . (mpmole) (/o>

0 5170±102 0.02114 0 10"5 6636 ±742 0.02713 -22 10"' 5939 ±685 0.02429 -11 10"3 2512±102 0.01027 40 10"2 30± 6 0.00012 99-100 " See Table 5.

Discussion Reed (2, 6) showed inhibition of the activity of amine oxidase in pea seedlings treated with B995. Oxidative deamination of tryptamine to indoleacetaldehyde, in the course of IAA formation, was demonstrated by Gordon and Nieva (7) in pineapple and by Clark and Mann (4) in the pea seedling. . The latter confirmed the presence of amine oxidase in the pea seedling. Thimann and Grochowska (8) concluded that pea stem tips probably convert tryptamine to IAA by means of a system having amine oxidase activity. By histochemically studying the pea seedling, amine oxidase activity was demonstrated to be high at the apical end of the epicotyl, decreasing toward the base (Plates 1, 2 and 3). Enzyme activity was highest in the cotyledon. Thimann and Skoog (9) reported that IAA was richer at the apical and of the pea seedling than at its base. Histochemical findings showed that monoamine oxidase inhibitors 558 M. KIMURA and C. TANAKA and B995 reduced amine oxidase activity in the pea seedling (Table 4). Hence, it is likely that inhibition of IAA formation due to inhibition of amine oxidase activity

is responsible for growth inhibition by B995 in pea seedlings. The fact that mono- Downloaded from https://academic.oup.com/pcp/article/12/4/551/1886246 by guest on 28 September 2021 amine oxidase inhibitors also inhibited germination, growth and the activity of amine oxidase in peas suggests a close relationship between the activity of amine oxidase and formation of IAA. If inhibition of amine oxidase leads to inhibition of IAA synthesis; hence, to inhibition of growth, then growth retardation could be reversed by treatment with IAA. Evidence is presented that growth suppression of pea and rice (Fig. 2) by safrazine was reversed by the addition of IAA to the culture solution. While the shoot growth in rice was suppressed by monoamine oxidase inhibitors, root growth was promoted by them. Audus (10) has shown that the optimal concentration of auxin for stimulation of root growth was only 1/100,000 that for shoot growth. The level of IAA reached after treatment with monoamine oxidase inhibitors may result in suppression of shoot growth but promotion of root growth. The authors wish to express their sincere thanks to Prof. Y. Tsukamoto and Prof. M, Fujiwara, Kyoto Univ. and Dr. K. Shimamoto, Takeda Co. Osaka for their continual encouragement and valuable advice throughout this work. They also grateful to Dr. Y. Masuda, Osaka City Univ. and Dr. H. Yamada, Kyoto Univ. for their valuable advice.

References

(7) Pletscher, A.: Monoamine oxidase inhibitors. Pharmacol. Rev. 18: 121-129 (1966). (2) Reed, J. D.: Tryptamine oxidation by extracts of pea seedlings: Effects of growth retardant /3-hydroxyethyl-hydrazine. Science 148: 1097-1099 (1965). (3) Glenner, G. G., H.J. Burtner and G. W. Brown, Jr.: The histochemical demonstration of monoamine oxidase activity by tetrazolium salts. J. Histochem. 8: 491-605 (1957). (4) Clark, A. J. and P. J. G. Mann: The oxidation of tryptamine to 3-indoleacetoaldehyde by plant amine oxidase. Biochem. J. 65: 763-774 (1957). (5) Wurtmann, R. J. and J. Axelrod: A sensitive and specific assay for the estimation of monoamine oxidase. Biochem. Pahrmacol. 12: 1439-1441 (1963). (6") Reed, J. D.,T. C. More and J. D.Anderson: Plant growth retardants B995: A possible mode of action. Science 148: 1469-1471 (1965). ( 7) Gordon, S. A. and F. S. Nieva: The biosynthesis of auxin in the vegetative pineapple II. The precursors of indoleacetic acid. Arch. Biochem. 20: 367-387 (1949). ( 8) Thimann, K. V. and M. Grochowska: The role of tryptophan and tryptamine as IAA precursors. Biochem. Physiol. Plant Growth Substances. Edited by F. Wightman and G. Setterfield. p. 231-242. 1968. ( 9 ) Thimann, K. V. and F. Skoog: On the inhibition of bud development and other functions of growth substance in Vida Faba. Proc. B. 114: 317-319 (1934). (10) Audus, L. J.: Physiology of growth retarding chemicals. Ann. Rev. Plant Physiol. 15: 271-302 (1964).