Sakai, Shingo

Simplified Chinese

Okayama University Scientific Achievement Repository

INTERACTION OF PLANT GROWTH REGULATORS IN AUXIN-INDUCED ETHYLENE PRODUCTION Shingo SAKAI Ethylene is produced bya number of plant ti岱 uesand a旺ectsmany physiological as well as bi∞hemical proces田sin plant ti岱 ues(1-3). 1 t has b知 1suggested that endogenous auxin level regulates endogenous ethylene production (4) and exogenously applied auxin stimulates ethylene production by inducing the enzymes involved in the formation of the gas (5-8). While ethylene production by plant tissue is greatly stimulated by auxin， ithas also shown that other plant growth regulators a旺ectethylene production. Bean stem田gmentsexcised from plants which had been sprayed with gibberellin produced ethylene twice the amount produced from unsprayed control plan也 (4). Kinetin at conantration of 10-8 to 10-. M stimulated ethylene production in etiolated伊aseedlings (9). Interaction of auxin， kinetin and gibberellin in ethylene production has b田nreported by Fuchs and Lieberman (9) with etiolated pea seedlings and stem 記gments. They have shown that in stem田gmen也 excisedfrom 6-day-old seedlings， ethylene production was considerably stimulated if kinetin was added with axuin. They have also shown that gib加rellinhad no e旺ecteven in the presence of auxin or kinetin. Those results clearly indi伺 tetha t ethy lene prod uction by plan t tissue is regulated by plant growth substances and there is an apparent interaction among them. Although many workers have inter，田tedin these phenomena， their attentions have mainly directed to physiological effects of ethylene on plant growth， and only a few biochemical studies on ethylene synth田ismodi自edby plant growth regulators have been reported. It is the purpose of the pre田ntpaper to elucidate the effects of plant growth regulato四 onauxin-induced ethylene productioIl. MATERIALS AND METHODS Plant matenal Etiolated 記edlingsof mungbean (Phaseolus aureus Roxb.) were grown， as previously described (7)， on a 0.4 ~ぢ agar-gel bed at 250C in the dark. After 72 hr， when hypocotyl length r，回ched30 to 35 mm， a 6 mm-long hypocotyl segment 加lowthe cotyledonary hook was excised from each seedling. lncubation Ten segments (0.14 g) were placed in a 25 ml-glass vial containing 122 S. Sakai 0.5 ml of an incubation medium. Fifty mM phosphate buffer， pH 6.8， containing 50 J.lg/ml chloramphenicol was used as incubation bu町er.Potassium indoleacetate was used as auxin. Plant growth regulators were dissolved in the above buffer solution. The glass vial was closed with a si1i∞n rubber stopper and incubated at 300C in the dark. After 12 hr incubation， the ethylene content in the gas phase was measured with a gas chromatograph. Assay 01 relative levels 01斤eeauxin in the tissue Twenty hy戸x::otyl田gmentswere incubated at 300C for 9 hr in a Petri dish (3 cm in diameter) with 1 ml of 0.1 mM Carboxyl-14C-IAA* (0.1μCi) in the incubation buffer with or without other plant growth regulators. At the end of the incubation period，配gmentswere thorough-ly washed with ice-chil1ed water and homogenized with 80 % ethanol in a glass homogenizer. The homogenate was centrifuged at 3.000 x g for 10 min. and precipitate was extracted with 80 % ethanol two more times. The combined ethanol extract was concentrated in vacuo to dryness， and the residue was dissolved in 1 ml of 80 % ethanol. The ethanol solution was then chromatographed on Toyo filter paper No. 51 with isopropanol-ammonium hydroxide-water (8: 1 : 1， v /v). Zones cor-responding to free IAA (Rf 0.61) and IAAsp紳 (Rf0.31) were cut out under UV light and radioactiviti田 inthese sections were measured with a liquid scintillation spectrometer. RESULTS Effect 01 auxin concentγαtion on ethylene Tγoduction Without auxin， mungbean hy卯∞tyl 田gments produced small amounts of ethylene. One μM auxin supplied in the incubation bu百erincreased ethylene production and as auxin concentration was raised， ethylene production progressively increased (Fig. 1). However， the re-spon句 ofsegments to auxin was maximal at 0.5 mM auxin. Effects 01 plant growth regulators 0ηωxin-induced ethylene prodl蹴 tionFig. 2 indicates that kinetin alone stimulated ethylene production though its e町ectwas not striking as the auxin effect. The effect of kinetin was also dependent upon kinetin∞n偲 ntrations. In the absence of added auxin， 0.1 mM kinetin increased ethylene production 10-fold during 12 hr. When 0.1 mM or lower of auxin was pre罰則， kinetin further stimulated auxin-indu白dethylene production， and a significant synergism of kinetin and auxin was observed. However， the magnitude of stimulation by kinetin to kinetin-frl田 controldecreased as auxin con偲 ntrationwas increased， and in the pre記 nceof 0.5 mM auxin; kinetin did not show any stimulative e百ect.市 IAA: Indoleacetic acid. 判 IAAsp: Indoleacetylaspartate. Interaction of Plant Growth Regulators in Ethylene Pr吋uction 123 6 0-0 ??』????? 『 「???』? ?????』 ?? ??↑?。7 6 5 4 3 -Log ( IAA) Fig. 1. Ethylene prodnction by hyp∞otyl民 gmentsindnced with varions con但ntrationof auxin (IAA). Ethylene production induced by auxin was also stimulated by gib-berellin (Fig. 3)， although gibberellin effect was smaller than kinetin efect. Presence of 0.1μM gibberellin stimulated about 30 % over control value， and no further stimulation was observed even higher concen-trations of gibberellin were used. As in the ca田 ofkinetin， 0.5 mM auxin induced ethylene production was not affected by gibberellin at any concentrations used. Gibbere1lin， however， did not clearly stimulate endo-genous ethylene production (Fig. 3). Fig. 4 indicat白 thatabscisic acid at concentration over 1μM inhibited auxin-induced ethylene production. Rate of inhibition of ethylene pro-duction at given abscisic acid ∞ncentration was independent upon auxin con白 ntration，thus， 0.01， 0.1 and 1 mM abscisic acid achieved about 15， 55 and 77 % inhibition， resp配tively，in the pr回enceeither 0.01， 0.1 and 0.5 mM auxin. Abscisic acid had litle effect on endogenous ethylene production (Fig. 4). Effects ofρlant growth regulators on endogenous levels of free J AA in the tissue It has bf:知1suggl由民dthat the rate of ethylene production by pea S. Sakai 124 』 ???????????」???? ュ? ????」? ? ? ?↑ ?IAA O.OlmM IAA 4 765 しーog(Kinetin) 8 Fig. 2. Stimulation by kinetin of auxin.induced ethylene pr，吋uctionby mungbean hyp∞otyl segments. stems after app1ication of auxin closely followed the levels of free auxin in the tissue (10). Since， inthe pre詑ntinvestigation t∞， it was∞n-sidered that auxin-induced ethylene production was regulated by endo. genous levels of IAA in the tissue， the relative levels of free IAA in the tissue after application of plant growth regulators were investigated. Although， kinetin and gibberel1in greatly stimulated ethylene production， the relative level of free IAA in the tissues treated by kinetin or gib-berellin was only s1ight1y higher than that of control tissue (Table 1). TABLE 1. Effects of plaot growth問思llators00 the level of fI田 IAAaod IAAsp fonnatioo io muogbeao hy似おotylsegmeots. IAAsp cpm 15.822 15.496 16.226 18.412 Free IAA cpm 3.410 4.356 4.298 6.018 Additions None Klnetin (0.1 mM) Gibbere1in (0.1 mM) A国cisicacid (0.1 mM) 125 Interaction of Plant Growth Regulators in Ethylene Production ?????』???? 『 ? ?? ?? ??」 ?? ? ? ?↑ ?0.5 mM <> 0 IAA o ~ a 一。mM 0.1 IAA 3 mM 4 0.01 765 しーog(GA) IAA Fig. 3. Stimulation by gibberellin (GAl of auxin-induced ethylene pr叫 uctionby mungbean hypocotyl segments. Treatment with abscisic acid reduced ethylene production (Fig. 4) but the trl回 tmentrather increased the relative level of endogenous IAA in the tissue and the formation of IAAsp. DISCUSSION The magnitude of the inductive increase in ethylene production is dependent on axuin concentration (Fig. 1). However， the effect of auxin was maximal at 0.5 mM. E旺ectsof plant growth regulators on auxin-induced ethylene pro~ duction as well as on relative free auxin level in the ti岱 uerevealed some interrelationships among actions of auxin and other plant growth regulators. The data with kinetin indicate that the e庄町tof kinetin is dependent upon auxin and maximal level of ethylene production appears to be regulated by auxin level. This is c1early shown in the prl白 enceof 0.5 mM auxin where kinetin does not show any stimulative e旺ect(Fig. 2). Capacity of ethylene production induced by 0.5 mM auxin 田emsto be maximal for the hypocotyl田gmentsand this is consistent with the fact that auxin level higher than 0.5 mM no further stimulation is observed (Fig. 1). Stimulation of endogenous ethylene production by S. Sakai ??????『???』?」???? 「 ?? ???」??? ?↑ ?0.5 mM 126 3 6 5 4 -Log(ABA) 7 Fig. 4. Effect of abscisic acid (ABA) on auxin-induced ethylene production by mungbean hyぴおotylsegments. kinetin a10ne may be considered to be synergistic ef，配tof kinetin with endogenous auxin. A1though kinetin treatment slightly increased re1ative endogenous auxin 1eve1 in the tissue， the great stimu1ative effect of kinetin on ethy1ene production was not exp1ained by the increase in re1ative endo-genous auxin 1evel. Therefore， itis considered that the stimu1ative effect of kinetin may be to magnify the auxin action. Abe1回 (5)showed that auxin-induced ethy1ene production by eti01ated bean hyぴlCoty1sections was inhibited by actinomycin D and to a 1esser extent by puromycin. Ethy1ene production by pea seedlings induced by auxin is a1so inhibited by cycloheximide and actinomycin D (6). We a1so showed that auxin-induced ethylene production by mungbean hypo-cotyl segments was inhibited by cycloheximide， puromycin and actin。mycin D (7，8). Those resu1ts suggest the inv01vement of RNA and protein synthesis in the induction of ethylene evolution by auxin. Since kinetin stimu1ates RNA synthesis (11，12) and kinetin e旺ect田emsto magnify the auxin action on induction pr∞e羽田， it can be assumed that Interaction of Plant Growth Regulator冨inEthylene Production 127 the effect of kinetin on ethylene production is resulted from the stimu-lation of RNA synthesis caused by auxin. In contrast to the experimental results with pea s田dlings(9)， gib-berellin stimulated auxin-induced ethylene production by mungbean hypocotyl田gments. Apparent effect of gibberellin is simi1ar to that of kinetin although degree of stimulation is not as great as kinetin and gibberellin effect was saturated at a lower concentration (Fig. 3). It has been suggested that gibberellin increa田sdiffusible auxin in the tissue (13). As shown in Table 1， gibberellin increased the relative free auxin level about 26 % in the presence of 0.1 mM auxin. This may account for 30 % incr伺 seof ethylene production by gibberellin in the pre田n白of 0.1 mM auxin (Fig. 3). If， however， gibberellin generally increases fr田 auxinlevel in tissue， endogenous ethylene production in mungbean hypocotyl should also be increased by gibberellin， which was not c1early observed. The author a田um回 thata slight stimulation of endogenous ethylene production by gibberellin was not detected due to the田nsitivityof the gas chromatograph. Auxin-induced ethylene production is inhibited by abscisic acid， and the inhibition is not competitive with auxin. This means that abscisic acid acts probably independent of auxin action. This assumption is further supported by the fact that abscisic acid inhibited ethylene pro-duction though it significant1y increa田dthe relative level of endogenous free IAA in the tissue (Table 1). A number of experiment indicated that abscisic acid inhibited RNA synthesis (14-16). Therefore， itis as-sumed that abscisic acid inhibited the synthesizing proce回 ofethylene producing system， presumably the synthesis of RNA. SUMMARY Auxin induced an increase in the rate of ethylene production by hypocotyl segments of etiolated mungbean seedlings. Kinetin stimulated auxin-induced ethylene production as well as endogenous ethylene pro-duction. Ethylene production induced by auxin was also stimulated by gibberellin， although gibberellin e庄町twas smaller than kinetin e町民t.Abscisic acid inhibited auxin-induced ethylene production， and the in-hibition is not competitive. ACKNOWLEDGEMENTS The author is deeply indebted to Dr. Hidemasa Imaseki. Nagoya University. for his cordial instruction and discussions throughout this study. He also expresses his thanks to Prof. lsamu Baba for his valuable advices to prepare this manuscrlpt. 128 S. Sakai REFERENCES L Burg. S. P. 1962. The physiology of ethylene formation. Ann. Rev. Plant Physio1. 13; 265-302. 2. Pratt. H. K. and Goeschl. J. D. 1969. Physiological roles of ethylene in plants. Ann. Rev. Plant Physiol. 20: 541-584. 3. Abe1es. F. B. 1972. Bi国 ynthesisand m配 hanismof action of ethylene. Ann. Rev. P1ant Physio1. 23: 259-292. 4. Abel回.F. B. and Rubinstein. B. 1鉛4. Regulation of ethy1ene evo1ution and leaf abscission by auxin. P1ant Physio1. 39:鋭)3-969.5. Abeles. F. B. 1966. Auxin stimulation of ethy1ene evo1utlon. P1ant Physio1. 41 : 585-5田.6. Burg. S. P. and Burg. E. A. 1969. Interaction of ethylene. oxygen and carbon dioxide in the control of fruit ripening. Qua1. P1ant. Mater. Veg. 14: 349-359. 7. Sakai. S. and Imaseki. H. 1971. Auxin-induced ethy1ene pr，吋uctionby mungbean hyp∞oty1 segments. P1ant Cel Physlo1. 12: 349-359. B. Sakai. S. 1973. Bi∞hemical studies of ethylene synthesis in plants. D∞tral dissertation. Nagoya University. 9. Fuchs. Y. and Lieberman. M. 19f:氾. Effects of kinetin. IAA and gibbeIちllinon ethylene production. and their interaction ln growth of seedling百・ P1ant Physio1. 43: 2029-2036. 10. Kang. B. G.. Newcomb. W. and Burg. S. P. 1971. Mechanism of auxin-induced ethy1ene production. P1ant Physio1. 47: 504-田9.1. Schneider. M. J. Lln. J. C. J. and Sk∞g. F. 1969. Nucleic acid metabolism durlng cytokinin induced cellular differentiation. Plant Physio1. 44: 1207-1210. 12. Sugiura. M.. Umemura. K. and Ota. S. 1962. The effect of kinetin on protein 1evel of toba∞o 1eaf disks Physio1. P1antarum 15: 457-必4.13. Kuraishi. S. and Muir. R. M. 1962. Increase in diffusib1e auxin after treatment with gibberel1in. Science 137: 760-761. 14. Pearson. J. A. and Wareing. P. F. 1969. Effect of a凶cislcacid on activity of chromatin. Nature 221: 672-673. 15. Vi1ie悶.T. A. 1968. An autoradiographlc study of the effect of p1ant hormone abscisic acid on nucleic acid and protein metabolism. P1anta B2: 342-354. 16. Walton. D. C..おofi.G. S. and Sondheimer. E. 1970. The ef，田tsof abscisic acid on growth and nuc1eic acid synthesis in excised embryonic bean ax田. Plant Physio1. 45: 37-40. 

INTERACTION OF PLANT GROWTH REGULATORS IN AUXIN-INDUCED ETHYLENE PRODUCTION

Abstract

Similar works

Full text

Available Versions

Okayama University Scientific Achievement Repository

Okayama University Digital Information Repository