Interaction of Growth-Retarding Compounds and Gibberellin on Indoleacetic Acid Oxidase and Peroxidase of Cucumber Seedlings" 2 Abraham H

Interaction of Growth-retarding Compounds and Gibberellin on Indoleacetic Acid Oxidase and Peroxidase of Cucumber Seedlings" 2 Abraham H. Halevy The Hebrew University, Faculty of Agriculture, Rehovot, Israel

Several groups of growth-retarding chemicals for each dish and 5 dishes for each treatment. have been reported in recent years. They belong to Dishes were covered immediately after sowing with quite distinct chemical classes, but their effect on several layers of black cloth, and placed at a constant plant growth is similar. These include nicotiniunm temperature of 250. After 94 hours, 10 seedlings compounds (31), quaternary ammonium carbamates were taken at random from each dish, measured, dis- (16), phosphonium compounds (37), choline ana- sected into cotyledons, radicles, and hypocotyl tips logues (42), and succinnamic acid derivatives (39). (upper 0.5 cm) and used for enzyme determinations. Unlike other types of growth inhibitors (e.g. maleic The chemicals used in these experiments were: hydrazide) these compounds retard stem elongation potassium gibberellate (GA), 2-isopropyl-4-dimethyl- without causing malformation of the plant. Their amino-5-methylphenyl-1-piperidinecarboxylate meth- effects on plants are in many aspects opposite to yl chloride (Amo-1618), an isomer of Amo-1618, 3- those of gibberellin (3,42). Most of these inverse isopropyl-4-dimethylamino-6-methylphenyl-1 -piperi- effects of the growth retardants and gibberellins are dine carboxylate methyl chloride (Carvadan) (pro- mutually antagonized when both chemicals are ap- vided by Dr. H. M. Cathey, Plant Industry Station, plied to the same plant (2, 3, 5, 7, 13, 14, 22, 27, 28, Beltsville), 2,4-dichlorobenzyl-tributylphosphonium 41). It was therefore suggested (27,42) that they chloride (Phosfon) (obtained from the Virginia an( be designated antigibberellins. This terminology Carolina Chemical Company), 2-chloroethyl trimeth- has caused controversy. While Lockhart (27) con- ylammonium chloride (CCC) (at gift of the Cyan- cluded that a functional interaction exists between amid International Company), N-dimethylamino suc- gibberellin and the growth retardants, Kuraishi and cinnamic acid (B-995) (obtained from Dr. J. A. Rid- Muir (25) came to the conclusion, that these com- dell, Naugatuck Chemical Div., U.S. Rubber Co.) pounds do not interact directly with gibberellin, but and maleic hydrazide (MH). rather with auxin. Stuart and Cathey (40) also felt Homogenates were made by blending 250 mg of that the growth-retarding chemicals should not be tissue from each organ in 10 ml of ice-chilled 0.1 M called antigibberellins. Our study presents data potassium phosphate, pH 6.1. The homogenates which are pertinent to this discussion. were filtered and immediately processed. Proper We have already shown that a growth-retarding dilutions were obtained using the buffer solution. substance (Amo-1618) increased peroxidase activity Results were calculated on a dry weight basis deter- in cucumber (13) and citrus (32) seedlings. Per- mined on parallel samples. oxidase is generally thought to participate in the Peroxidase determinations were made according IAA-oxidase system (38). We have suggested to McCune (29), using guaiacol as hydrogen donor. (13) that the growth retardants may affect the auxin The formation of tetraguaiacol was followed at 47C level of plant tissues by enhancing auxin destruc- mA in a spectrophotometer (Spectronic 20, Bausch tion. The present study gives support to this hypo- and Lomb). Activity was measured by increase in thesis. optical density (OD) from 60 to 90 seconds. The rate was found to be linear during this time period. Materials and Methods Activity is expressed as A OD in 60 to 90 second period per 1 mg of dry weight. Cucumber seeds (Cucumis sativus L. var Beit The oxidation of IAA was measured at 525 m,u Alpha, Hazera Seed Co., Tel Aviv) were sown in 9 using the Bausch and Lomb Spectronic 20 spectro- cm petri dishes on 2 layers of Whatman No. 2 filter photometer. The Salkowski reagent used was as paper, which had been saturated with water or the modified by Pilet (35). Reaction mixtures were indicated chemical solution. Fifteen seeds were used prepared according to the method of Goldacre et al. (12) and consisted of 10-4 M 2,4-dichlorophenol, 10-4 MnCl9, 2 x 10-4 M IAA and 1 ml of enzyme 3Received April 29, 1963. solution to 10 ml of 0.1 M potassium phosphate pH 2 Contribution from The National and University In- 6.1 added in stitute of Agriculture, Rehovot, Israel. 1963 Series, this order. Optical densities were con- 575-E. verted to ug of residual IAA using a curve prepared 731 732 PLANT PHYSIOLOGY

J _

--e~ 'l ~- w _ _-- _-4. PEROXIDASE (COT) II 10-i 10 10 1O02 D-5 10-1104 10-3 GIBSERELLIN (M) AMO-1618 (M) FIG. 1. Effects of various concentrations of gibberellin on elongation of hypocotyl and relative peroxidase and IAA-oxidase activity in cotyledons and hypocotyl-tips of cucumber seedlings. Standard errors for each curve are drawn as vertical bars. Peroxidase values for water control of cotyledons and hypocotyl are respectively 102 and 141 A OD in 60 to 90 seconds period/i mg dry weight. IAA-oxidase values for water control of cotyledons and hypocotyl are respectively 165 and 189 gg IAA metabolized/mg dry weight hour. Length of hypocotyl in water was 40.5 mm. FIG. 2. Effects of various concentrations of Amo-1618 on elongation of hypocotyl and relative peroxidase and IAA-oxidase activity in cotyledons and hypocotyl-tips of cucumber seedlings. Standard errors for each curve are drawn as vertical bars. Values for water controls are as those stated in legend to figure 1. by plotting the optical densities of standards. Ac- Table I tivity is expressed as the disappearance of Ag IAA/ Effects of Variouis Concentrations of Gibberellic Acid nit mg dry weight hour. Activity of Peroxidase and IAA-Oxidase in Radicles of Cucumber Seedlings Figures in each column followed by the same letter Results are not significantly different. Figure 1 shows the effect of various concentra- Concentration of GA Peroxidase IAA-Oxidase tions of GA on elongation of the hypocotyl and en- (M) (A OD)* ** zymic activity in cotyledons and hypocotyl tips. It Olg)

is obvious that an inverse relation exists between 0 92 a 123 a elongation and enzymic activity. The correlation 2.5 x 10-5 97 a 119 a between the effect of GA on hypocotyl growth and 10-4 87 a 90 b 2.5 x 10-4 84 a 75 b its effect on peroxidase and IAA oxidase in this or- 7.5 x 10-4 80 a 82 b = gan was found to be highly significant (r 10-3 83 a 68 b -0.620** and r = -0.735**, respectively). GA also 2.5 x 10-3 84 a 76 b inhibits enzyme activity in the cotyledons, but to a 5 x 10-3 79 a 80 b lesser extent. The reduction is proportional to the * A OD in 60 to 90 second/i mg dry weight. concentration of the reagent (fig 1). GA had no ** ,ug IAA/mg dry weight hour. HALEVY-GROWTH-RETARDING CHEMICALS AND GIBBERELLIN 733 Table II Effects of Gibberellic Acid and Amo-1618 on Growth and Peroxidase Activity in Various Organs of Cucumber Seedlings Figures in each column followed by the same letter are not significantly different.

Treatment Average Length (mmn) Peroxidase Activity (A OD)* Hypocotyl Radicle Hypocotyl Radicle Cotyledons H20 39.2 b 50.8 a 130 b 81 b 90 b GA (7 x 10-4M) 62.7 a 48.3 a 71 c 66 b 68 c Amo-1618 (1.5 x 10-3M) 20.1 c 21.0 b 226 a 148 a 130 a GA (7 x 10-4 M) + Amo-1618 (1.5 x 10-8M) 40.3 b 19.9 b 146 b 143 a 85 b * A OD in 60 to 90 second/mg dry weight. significant effect on peroxidase activity of the radi- chemical but to its retardation effect on radicle cles, but inhibited IAA-oxidase activity (table I). growth (fig 3). It should be noted that GA did not stimulate radicle The interaction of GA and Amo-1618 on enzymic growth at this range of concentration (table II, see activity was studied at concentrations nullifying their also ref. 15, table II). inverse effect on hypocotyl length (14), and are The effect of various concentrations of Amo- summarized in tables II and III. 1618 on hypocotyl growth and enzymic activity in The inverse effects of GA and Amo-1618 on the cotyledons and hypocotyl tips is presented in figure activity of both enzymes in hypocotyl and cotyledons 2. Here again high correlation was found between were nullified by each other (tables II and III). the effect of the various concentrations of the chem- With respect to the radicle, a partial interaction be- ical on retardation of hypocatyl growth and its ef- tween GA and Amo-1618 was apparent in their effect on stimulating activity of peroxidase and IAA- fects on IAA-oxidase activity (table III), but peroxi- oxidase in hypocotyl tips (r = -0.635** and r = dase was unaffected (table II). -0.689**, respectively). A similar but lesser effect To test whether the stimulating effect of Amo- was detected on enzymic activity in the cotyledons. 1618 on peroxidase and IAA-oxidase is common to The effect of Amo-1618 on radicle growth and other growth-retarding substances, the comparative enzymic activity is shown in figure 3. Retardation effect of 5 growth-retardants and one growth in- of radicle elongation began at a higher concentration hibitor (maleic hydrazide) on IAA oxidase and than that affecting hypocotyl growth (3 X 10-4 M peroxidase activity was studied (table IV). For com- compared to 2.5 x 10-5 M) (fig 2). However, at parison between the various reagents a concentration the high concentrations of Amo-1618 (from 10-3 M of each compound was chosen which reduced elonga- on), the relative inhibition of radicle growth was tion of the hypocotyl to about 50 % of that of water even stronger than that of hypocotyl. The effect of control. This concentration was determined in pre- Amo-1618 on peroxidase activity was related to its liminary experiments. The effect on the radicle concentration, but the effect on IAA-oxidase was varied according to the substance applied (table IV). negatively correlated not to the concentration of the B-995 did not retard radicle growth significantly

Table III Effects of Gibberellic Acid and Amo-1618 on IAA-Oxidase Activity in Various Organs of Cucumber Seedlings Figures in each column followed by the same letter are not statistically different.

Treatment ,ug IAA/mg dry wt hr Hypocotyl Radicle Cotyledons H20 174 b 138 c 156 b GA (7 X 10 -4M) 63 c 90 d 110 c Amo-1618 (1.5 x 10-3M) 421 a 385 a 309 a GA (7 X 10 -4 M) + Amo-1618 (1.5 X 10-3M) 160 b 168 b 163 b 734 PLANT PHYSIOLOGY while Phosfon and MH inhibited radicle elongation much more than that of hypocotyl. The data in table IV show that the activity of both enzymes was stimulated by all 5 growth-retarding compounds, but not by maleic hydrazide. How- ELONGATION- 60 ever, the increase in enzymic activity was different I40 even in the hypocotyl where the retardatioin effect was similar. CCC and Phosfon were the nmost active in promoting enzyme activity, and B-995 the on wvas 20~ least active. The effect the radicle generally the strongest, and on the cotyledons the N-eakest. It should be noted that except for one case (B-995, Io radicle), the relative increase in IAA-oxidlase activity was higher than of peroxidase. 300_ IAA OXIDASE Discussion

The present paper shows a response to artificial 200 dwarfing by means of growth-retarding comiipounds. tuI~~~~~~ ~ ~ It has been reported that dwarf varieties of bean Xb PEROXIDASE (20), corn, and pea (30) plants have a greater peroxidase activity than the normal varieties of the the .100 same species. Treatment with GA increase growth rate of these dwarfs, and decrease the peroxidase (29, 30) and IAA-oxidase level (4) of the 0ffi$$ 1I 5 1_-2 Or 14 V -3 dwarfs. Decreased IAA oxidase level in normal AMO-1618 (M) GA-treated plants has been reported by several FIG. 3. Effects of various concentrations of Amo- workers (4,9, 19,). In the present work inhlibition 1618 on elongation and relative peroxidase and IAA- of growth by means of growth retardants was asso- oxidase activity of radicles of cucumber seedlings. ciated with increased peroxidase and IAA-oxidase Standard errors for each curve are drawn as vertical activity. In addition, the restoration of grow-tlh to bars. Peroxidase value for water control is 80 A OD in the normal rate by treatment with GA reducedl en- mg weight. IAA-oxidase 60 to 90 seconds period/i dry seedlings. value for water control is 138 mg IAA metabolized/mg zymic activity to about that of untreated dry weight hour. Length of radicle in water was 51.6 It was found in this study that not only activity of mm. the enzymes was inhibited by GA an(d promlote(d by

Table IV Effects of Growth Retarding Chemicals at Concentrations Cauising 50 % Decrease in Hypocotyl Length on Activity of Peroxidase and IAA-Oxidase in Various Organts of Cutcumber Seedlinigs Figures in each column followed by the same letter are not statistically different. Average length (mm) Hypocotyl Radicle Cotyledons Treatment Hypocotyl Radicle Peroxidase* IAA- Peroxidase* IAA- Peroxidase IAA- oxidase** oxidase** oxidase**

H20 39.6 a 50.1 a 109 d 160 e 72 d 131 d 86 c 140 d Amo-1618 (1.5 x 10-3 M) 19.8 b 21.7 c 177 bc 370 c 138 bc 375 b 119 b 276 b Carvadan (2 X 10-3 M) 20.0 b 33.1 b 163 bc 270 cd 98 cd 284 bc 109 bc 216 c CCC (7 x 10-3 M) 20.1 b 27.4 bc 259 a 525 b 189 a 567 a 131 b 290 b Phosfon (1.7 X 10-4 M) 19.7 b 12.6 d 212 ab 644 a 145 b 457 b 173 a 459 a B-995 (3 X 10 -3 M) 19.9 b 49.2 a 150 c 234 d 113 c 169 d 101 c 182 c MH (10-3 M) 20.2 b 15.3 d 111 d 175 e 75 d 146 d 77 c 136 d

* A OD in 60 to 90 second/mg dry wt. ** ,ug IAA destruction/mg dry wt hour. HALEVY-GROWTH-RETARDING CHEMICALS AND GIBBERELLIN 735 Amo-1618, but that the intensity of inhibition or hibition effect. This can be evaluated from compar- promotion was inversely proportional to the effect of ing the effects of the growth retardants on enzyme the various concentrations of the chemicals on growth. activity to those of MH. Despite the strong inhibi- The experiments reported here were carried out tion of growth by MH, the effect of peroxidase ac- with seedlings grown in complete darkness to avoid tivity and auxin destruction was insignificant (table the possible interaction of light with the effect of the IV). The results of the effect of MH on IAA- growth regulators (7, 14, 15, 16, 26) or with the ac- oxidase are in accordance with those of Pilet (36). tivity of the enzymes (17). However, similar re- It thus seems that MH inhibits growth by acting else- sults with peroxidase were detected also with light- where. Lockhart (27) drew a similar conclusion grown see(dlings (13). Our results are not in agree- from his kinetic studies of growth. ment with those of Housley and Deverall (19), who The opposite effects of Amo-1618 and GA on found an effect of GA on IAA-oxidase in light- peroxidase and IAA-oxidase were mutually antagon- grown pea seedlings, but little or no response in ized when both substances were applied to the seed- dark. lings (tables II and III). An interaction of GA and The similarity in the effect of the chemicals on growth-retarding chemicals had been previously found the 2 enzymes may be explained by the fact that in some other processes such as stem and leaf elonga- peroxidase is considered to be a part of the enzyme tion (2, 3, 7, 14, 22,27,28,41), Ulothrix growth (5), system responsible for the destruction of IAA in vivo flowering (2,), flower sex expression (43), and (38,18). The direction of the effect of the growth catalase activity (13). The growth-retarding chem- regulators on peroxidase and IAA-oxidase was in- icals had therefore been designated as antigibberel- deed the same, but the degree of the effect was not. lins. All the growth-retarding compounds available The influence of both GA and the growth retardants at present are synthetic materials and there is no on IAA-oxidase was, in almost all cases, stronger evidence that these compounds occur naturally in than on peroxidase. This may be explained by the plants. However, some unidentified antagonists of fact that plant tissues contain several peroxidases, GA had been extracted and isolated from immature differing in their capacity to participate in the IAA- fruits of Ceratonia siliqua (6). oxidase system (11, 29) and, therefore, to respond The designation of antigibberellins to the growth to the growth regulators. retardants was considered unjustified by Stuart and It has been demonstrated that treatment with GA Cathey (40) and Kuraishi and Muir (25). Never- resulted in an increased level of endogenous auxin in theless, Lockhart (27) came to the conclusion that several plants (21, 24, 34). IAA oxidase activity is both Phosfon and CCC show complete interaction dependent on phenolic cofactors and inhibitors, some with gibberellin and should be called antigibberellins. of which have been recently isolated and identified Our results support Lockhart's assumption that the (8,33). Galston (9) reported that GA treatment in- growth-retarding substances are actually antigib- creased the content of the then unidentified phenolic berellins. We also agree with Lockhart's approach inhibitor of IAA-oxidase. that designation of these substances as antigibberel- All these data support the hypothesis suggested lins does not necessarily mean that there is any direct by Galston and Warburg (10) that the increase in competition or interference with biosynthesis but diffusible auxin resulting from GA treatment is prob- rather any interference with one of a few factors in ably mediated by an auxin oxidase inhibitor. This a catanary growth system. This interpretation may flavonoid inhibitor might be identical with the third abolish the reason for the lack of acceptance of anti- factor in the GA-auxin interaction, suggested by gibberellin designation to the growth retardants by Brian and Hemming (1). Kuraishi and Muir (25). They found (25) that It was suggested earlier (13) that the growth- CCC interacts more with IAA than with GA. If retarding chemicals may exert their effect by lower- we assume that auxin level is a factor in the activity ing auxin content of plant tissues through enhanced of both gibberellin (23) and the growth retardants, auxin destruction. Increased IAA-oxidase activity their interaction might thus be not direct, but through in seedlings treated with the various growth retardants their effect on IAA-oxidase or its cofactors and in- is demonstrated in the present work; related to this hibitors. is the recent report by Kuraishi and Muir (25) that a decrease in diffusible auxin follows CCC treatment. As with gibberellin, the effect of the growth- Summary retarding compounds on auxin destruction may pre- sumably be mediated by the level of the flavanoid co- Cucumber seedlings were grown in darkness on factors and inhibitors of the enzyme. However. no filter paper saturated with water, potassium gibberel- evidential support to this assumption has yet been late (GA), 5 growth-retarding chemicals :(2-iso- presented. propyl-4-dimethylamino-5-methylphenyl-1-piperidine- The effect of the growth-retarding substances on carboxylate methyl chloride (Amo-1618), 3-isopro- peroxidase and IAA-oxidase found in the present pyl-4-dimethylamino-6-methylphenyl- 1 -piperidinecar- study are similar to those of genetic dwarfing (4, boxylate methyl chloride (Carvadan), 2,4-dichloro- 20), and do not stem merely from the growth-in- benzyltributylphosphonium chloride (Phosfon), 2- 736 PLANT PHYSIOLOGY chloroethyl trimethylammonium chloride (CCC), and 9. GALSTON, A. W. 1959. Studies on IAA oxidase N-dimethylamino succinnamic acid (B-995) maleic inhibitor and its relation to photomorphogenesis. hydrazide, or both GA and Amo-1618. The effects pp. 137-57. In: Photoperiodism and Related of the chemicals on the activity of peroxidase and in- Phenomena in Plants and Animals. R. B. With- row, ed. AAAS, Washington, D. C. doleacetic acid-oxidase were studied in cotyledons, A. WV. AND H. 1959. An and 10. GALSTON, WARBURG. radicles, hypocotyl-tips. analysis of auxin-gibberellin interaction in pea GA inhibited alnd Amo-1618 stimulated the ac- stem tissue. Plant Physiol. 34: 16-22. tivity of peroxidase and IAA-oxidase in hypocotyl-tips 11. GARAY, A. S. AND F. MIHALY. 1959. Papierelek- and cotyledons. The effect was inversely correlat- trophoretische Trennung Peroxydase-wirkender ed with the effect of the various concentrations of the Agenten aus Pflainzen. Phyton 13: 55-7. chemicals on hypocotyl elongation. The same rela- 12. GOLDACRE, P. L., A. W. GALSTON, AND R. L. WEIN- TRAUB. 1953. The effect of substituted phenols tion was apparent xwith the effect of Amo-1618 on on the activity of the IAA oxidase of etiolated radicles. GA did not affect peroxidase activity in peas. Arch. Biochem. Biophys. 43: 358-73. radicles. and slightly decreased their IAA-oxidase 13. HALEVY, A. H. 1962a. Inverse effect of gibberel- activitv. lin and Amo-1618 on growth, catalase, and peroxi- The inverse effects of GA and Amo-1618 on per- dase activity in cucumber seedlings. Experientia oxidase and IAA-oxidase activity were mutually an- 18: 74-6. tagonized when both chemicals were applied at con- 14. HALEVY, A. H. 1962b. Interaction between gib- cenitrations nullifying their opposite effects on hypo- berellin and quaternary ammonium carbamates in cotyl growth. growth of cucumber seedlings. Bull. Res. Counc. Israel liD, 83-90. Stimulationi of peroxidase and IAA-oxidase ac- 15. HALVEY, A. H. AND H. M. CATHEY. 1960a. Effect tivity was found with all five growth-retarding com- of concentration and structure of gibberellins on pounds tested, but not with maleic hydrazide. growth of cucumber (cuicuiiiiis sativuts) seedlings. It is proposed that the growth-retarding chemicals Botan. Gaz. 122: 63-67. exert their effect on plant growth by interacting with 16. HALEVY, A. H. AND H. M. CATHEY. 1960b. Effect gibberellin in IAA-oxidase (or its cofactors and in- of structure and concentration of some quaternary hibitors). and( thlus affect the auxin level of tissues. ammonium compounds on growth of cucumber seedlings. Bot. Gaz. 112: 151-54. 17. HILLMAN, W. S. AND A. W. GALSTON. 1957. In- Literature Cited ductive control of IAA oxidase activity by red and near infrared light. Plant Physiol. 32: 129- 1. BRIAN, P. W. AND H. G. HEMMING. 1958. Com- 35. plementary action of gibberellic acid and auxins 18. HINMAN, R. L. AND P. FROST. 1961. A model in pea internode extension. Ann. Botany 22: 1-17. chemical systemii for the study of the oxidation of 2. CATHEY, H. M. 1959. Effects of gibberellin and indole-3-acetic acid by peroxidase. pp. 205-16. 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Gaz. 121: 233-37. diffusible auxin after treatment with gibberellin. 8. 1FURUYA, M., A. W. GALSTON, AND B. B. STOWE. Science 137: 760-61. 1962. Isolation from peas of co-factors and in- 25. KURAISHI, S. AND R. M. MUIR. 1963. Mode of hibitors of indole-3-acetic acid oxidase. Nature action of growth retarding chemicals. Plant 193: 456-57. Physiol. 38: 19-24. HALEVY-GROWTH-RETARDING CHEMICALS AND GIBBERELLIN 737 26. LOCKHART, J. A. 1961. Interaction between gib- 35. PILET, P. E. 1957. Dosage photocolorimiietrique de berellin and various environmental factors on stem l'acide 3-indoly-acetique: application a l'etude des growth. Amer. J. Botany 48: 516-25. auxines-oxidases. Rev. Gen. Botan. 64: 106-22. 27. LOCKHART, J. A. 1962. Kinetic studies of certain 36. PILET, P. E. 1957. Action of nialeic hydrazide on antigibberellins. Plant Physiol. 37: 759-64. in vivo auxin destruction. Physiol. Plant 10: 28. MARGARA, J. 1961. 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