Ethylene-An Unusual Plant Hormone Eric W. Ainscough, Andrew M. Brodie, and Anna L. Wallace Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand
Ethylene is a fascinating plant hormone that plays a critical role in almost every phase of development in many plants, including germination, growth, sex expression, the blooming of flowers, fruit ripening, aging, and leaf loss (16). It also functions in response to a whole series of stress situations such as wounding, low or high temperature, drought, water-logging, and the introduction of certain In this article we discuss some of the early chemicals (7,8). historical observations about this hormone, the production and concentration of ethylene in plants, the ethylene biosynthesis pathway, and the possible site of ethylene action.
Production and Concentration of Ethylene In Plants Ethylene is produced naturally by all higher plants and pmbably lower green plants, and algae. There appears to be no evidence that normal animal cells possess a biosynthetic pathway to ethylene. With the advent of gas chromatography an easy and sensitive method to analyze ethylene became possible, and this work was published in 1959 (14,151. From such work it was found that the concentrations of ethylene range from a few parts per million in nongrowing tissues to over 2000 ppm in a ripening fruit. These measurements were of ethylene gas found in the intercellular air spaces. The ethylene that has a function in growth control is that within plant cells (within the cytosol), and there is almost no information on its com~artmentedconcentrations within the cell or how it is distributed,
Some Early ObSe~ationSabout the Existence of a Hormone The long history of ethylene research in plant science has been described by Burg (9), Abeles (11, and Osborne (10); hence, only a brief summary is given here. Biblical texts from the Old Testament inform us that the prophet Amos gathered fruits of the fig tree. To hasten their ripening it was common practice to wound them by slashing them. In tropical countries it is agricultural folklore that the smoke from bontires would accelerate the ripening of fruits. The first scientific observation was reported to be made by Girardin in 1864 (11)when he observed the shedding of leaves from trees that were near to leaking gas mains. However, it was not until 1901 that Neljubow (12) discovered that the active principle in coal gas that produced such remarkable effects in plants was the simple two-carbon volatile hydrocarbon, ethylene, (also called ethene, CzH4).He showed a 'triple response' of pea plants grown in the dark when they were exposed to different concentrations of the gas. Elongated growth was arrested (0.1 pL L-'), lateral swelling of elongating parts occurred (1fi L-'1, and the normal direction of shoot growth was lost (10 pL L-'1. By the 1930's most of the known physiological effects in plants that could be induced by ethylene were described. The use of ethylene as a commercial fruit ripening agent for bananas, citrus, and other fruits had now been established. indole -3-aceticacid Plant physiologists, particularly from the Boyce wounding Thompson Institute, also observed that one ripe apple enclosed with pea plants prepared as before, could induce in them Neljubow's "triple response" and a single ripe fruit would speed up the ripening of other fruits enclosed with it. The suspicion that plants 1 - aminocyclopmpane themselves actually produce ethylene was confirmed carboxyiic acid (ACC) by Gane 1934 (13) using chemical absorptive techniques to remove the gases from ripening apples. Because of its effects on plant growth and development, Ethylene Forming Enzyme it was reluctantly accepted that ethylene was a plant hormone. Another interesting implication was that / O2 whereas other biological systems are in general subject to their own endogenously produced hormones, CO, + HCN plants are subject to the vagaries of ethylene present in their external environment in addition to their own output. Plants may be exposed to ethylene present in coal gas (10 ppm), in smoke (4 ppm), in car exhaust fumes (400 porn), in town air (10-50 .pub).. and in rural air (5 ipb). Figure 1. Ethylene biosynthesis pathway of higher plants.
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by auxin and has a short half-life (25-30 min). It is also subject to modification by wounding, stress, and red light. The ethylene forming enzyme is regulated by light, temperature, 0 2 and C02. Further mechanistic detail was obtained by feeding mung beans with a 1:l mixture of tetraorotio- and tetradeuterio-ACC and analvzine ~ ~ the ~ bead space by gas chromatopaphy and massspectrometry (241. The observed ratiu uf isotopomers rC2& and C2D,) = 0.990 showing a lack of an isotope effect. revealed i k ~ k w Similarlv. t h e same mixture was converted to i t s tetrabutyl ammonium salt and oxidized electrochemically and the observed isotooe rabo ( k d k , , )= 1.005. nlso showing the lack of an isotope effect. & k t i a l oneelectron oxidation of ACC a t the NH3' group was proposed to be the rate-determining step. Ring opening of the m i n e radical cation is then suggested to produce ethylene (Fig. 2). ~
~
-
~~~~
The Ethylene Binding Site I t has been found that other unsaturated molecules such as propene, 1-butene, acetylene, and carbon monoxide will compete with ethylene and will nroduce the ethvlene effect althbugh higher-concentratio& are required. However, saturated molecules such as ethane, propane, and 2-butene have no detectable effect (1,3).The site of etbylene action is not known, but the structural reauirements for biologicd activity of alkenes, acetylenes, and carbon monoxide suggest that a metal ion is present a t the ethylene receptor-site (1.21.This suggestion is supported by the inhibition of ethylene binding either by dithiocarhonate ions (25I or silver nitrate (26)in a cell-free svstem isolated from &&beans. Other studies have shown 2,5-norbornadi&1e is an antagonist of ethylene action (27). Administration of an anionic silver thiosulfate complex prevents cut carnations fromwiltine (28). Presumablv. the anionic comolex inhibits ethylene action by interf&ng with the bin&ng sites of ethvlene. These considerations have led to the oroposal that copper(1) ion is a t the ethylene binding site
a
Some Copper(1)-Ethylene Complexes I n one study the ligands bydrotris(3,5-dimethyl-1pyrazoly1)borate (HB(3,5-Me2pz)3) a n d hydrotris(1pyrazolyl) borate (HBpz3) (Fig. 1)were used as they proFigure 2. A mechanistic proposal for the formation of ethylene from ACC.
The Biosynthetie Pathway to Ethylene and the EthyleneAuxin Link Clear confirmation by Morgan and Hall (16, 17)that sprays of auxin (indole-3-acetic acid) increased the production of ethylene manyfold (in a n hour) was the next important discovery in the area. This was followed by the observation t h a t exposure of plants to etbylene led to a reduction in the flux of auxin transport (18).Ethylene production can be altered by response to a variety of stress conditions such as beat, cold, water-logging, mechanical perturbation etc. Other imnortant breakthrouehs came when it was established that'the two carbons ofithylene were derived from the number 3 and 4 carbons of the amino acid methionine (19) and t h a t S-adenosyl methionine (SAM) and 1aminocyclopropane-1-carboxylicacid (ACC) (20, 21) were intermediates in the biosyntbesis pathway from methionine to ethylene (Fig. 1). It is known that SAM and ACC are not active as hormonal substances. ACC is readily mobile and moves in the xylem (woody tissue). The conversion of SAM to ACC requires the use of an enzyme called ACC synthetase as was demonstrated in 1979 (22,231. The enzyme's activity is controlled (induced) 316
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Figure 3. The structures of HB(3,bMe2pz),and HBpz,.
~
.
=
vide imidazole-like ligands that are eommonly associated with copper(1) in nature (29).No structural data are available yet on the proposed ethylene binding site. The interaction of CuI and potassium hydrotris(3,5dimethyl-l-pyrazo1yl)boratein CHzClz in an ethylene atmosphere produces t h e neutral complex [Cu(B(3,5Me2pz)J(C2Hd1 t h a t is monomeric. The copper ion is coordinated to a n ethylene molecule and to a nitrogen atom from each of the three 3,5-dimethylpyrazole rings. The complex is essentially trigonally symmetric around the Cu-B axis (Fig. 4). Similarly, in the complex Cu(Bpz3XCzH&CuCl (Fig. 5) prepared as above but from two moles of CuCl and one of the Bpzs ion, one copper ion is bonded to two pyrazole groups and to an ethylene molecule in a trigonal-planar
(C2H4)]. Figure 4. The structure of [C~(B(3.5-Me~pz)~
O2 and H20. The plant then responds to the production of the diol(l8). Further studies are obviously required here. Future Prospects Details on the ethylene binding site need to be understood and the role ethylene plays in the important physioloeical events such as eermination. flowerine. fruit rioenin> etc. need to be eluc;dated. ~ e n e ' c l o n morall ~ the genes involved in ethvlene biosvnthesis will be carried out. the shelf life of fruits and vegetables Efforts to will continue. CSIRO in Australia has developed a number of plastic wrapping films that absorb ethylene, and this is the first step in developing an anti-aging material. Many fruits give off ethylene when ripening, and this (as well as the presence of extraneous ethylene) can result in aging and loss of freshness. On growing plants, the petals either fade or fall offand the leaves fall off. CSIRO has developed and patented a compound that removes ethylene from the atmosphere around plants. This pink-colored compound is incorporated easily into plastic films, and when ethylene diffuses into the film it eventually becomes wlorless. The pink coloration can be used as a n indicator of the degree of reaction. The ethylene scavenging fdm is best considered as part of the strategy required for extending shelf life. The range of applications of this pink compound bas not been explored yet. Figure 6 shows (on the left) carnations placed in a flask containing the ethylene scavenging compound (scrubber) in silicone, while (on the right) a control where the scrubber is absent. Both sets of carnations were treated with ethylene injections to promote aging. The treated carnations survived three or four days after the others had wilted. Further studies in this area will wntinue.
arrangement. The pyrazole ring of the Bpzs ion wordinates to a Cu-Cl p u p which is weakly associated with the Cu-Cl of a neighboring molecule. I n another study the copper(1) complexes [Cu(phen)LIC104(L= ethylene, acetylene, and CO) have been synthesized and the crystal structures determined from X-ray diffraction (30).
Figure 6. The effectof an ethylene scavenger on the survivai of two Sets of carnations. Figure 5. The structure of Cu(Bpz3)(C2H4)CuCI. In all these complexes the C-C bond distance is essentially the same as observed for the free molecule. Sigma bonding is suggested to be the dominant interaction between the ethylene and metal. These studies demonstrate that the coordination chemistry of copper(1)-monoalkenecomplexes is consistent with the proposed role of copper at the ethylene receptor site of plants. The wmpounds are stable to loss of alkene. The hydrophobic ethylene binding site appears to be asociated with Dolvoe~tideswithin the intracellular membranes (28,. 1; ha: dwn postulated that the bound ethylene is mnvened t o 1,2-dihydroxycthancin the presence of
Acknowledgment We thank Bob Holland CSIRO, Sydney, for information on the ethylene scavenging film as well as the photograph of the carnations. Literature Cited 1. Abeles, F B.Efhyhne inPlont B1ology;Aeademie Resa: N w York,1973. Chapter 3. 2. Burg, S. P.; Burg,E. A P l o n f Physiol. 1967,42, 1M. 3. Leshem Y The Mokculor and Horrnoml Bosls of Plonf Gmlvlh Rogukzfion; Permagon Press: Oxford. 1913, pp 141-143. 4. Imaseki, H. Inchemisfry ofPIonfHormnos;T&shashi, H . , Ed. ;C.R.C.Press:New York,1986, pp 24S264. 5. B W ~ , SP. P W . N ~ ~ . Asa., C ~U .S A . ism, 70, 591. 6. Cookson, C.; Oshme, D.J. Plonlo 1978, 144.39. 7. Yang, S. F; Hoffman, N. E.Ann. Rou. Plant Physiol. 1984.35. 155. 8. Rrady,C. J.Ann. Re". Plant Physio2. 1981.38.155.
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9. B w , S. P A m . Rev. PIantPhyaiol. lB69,13,265. 10. ajborne, D.J. In P h y m h o m m and &ad C o v o u n d r : A Comprehmsiur hI&, ~ 1 . 2 ~ : ~ tD hS.; Cmdrsm, ~ , P B.; H i e m , T. J. V., Eds: ElaevierNmth Holland:Amaterdam, 1918,265. 11. Girardin. J. L. J a h m b . Agr. lW4,7,199. 12. Neljubow, D.&A Bat. Centmlbl 11801,IO.128. 13. Gane, R. Nature 1984,134,1008. 14. Burg, S. P.;Stoluljk, J. A. J. B i o e h m Miembid Ihchnd. EM. 1958,1,246. 15. Huelin, F.E.;Kennetf B. H. Ndure 11)68.284,996. 16. Morgan, P.W.;Hall, W. C.Physid. P l a n f a r m 1962. 16.420. 17. Morgan. P.W.:Hall, W. C.Nohrre l W ,201,99. 18. Beyer, E.M. Jr;Mowan, P W. Plant Physlol. lB69.44.1690. 19. Liebeman. M.;Kunishi, A. T.; Mapon. h W.; Wardale, D. A PhntPhysioL 1888, 41,376. 20. A~z-, D.0.; yang, s E p h n f ~ h ~ ~1 i9 n d 60, . 891.
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21. A d a m , D. O.;Yang, S. F Pme Notl.Amd Sci., USA 1918,76,170. 22. Yu, Y B.; Adama, D.O.:Ymg, S. F.Amh. Bioeham. Bhphys. 1918,198,280. 23. B O N ~ ~ , H-~.R. T.; c.:~ e n d eH, P h n a 1918.145,293. 24. Pirrung, M. C.; MeGeehan, G. M . J h r Cham. Soe, l 9 W 208,5641. 25. Si4er.E. G.: Isenhour, E.M.Plrmt Physiol.,Suppl. 1981,67,52. 26. Sider, E. C. J. Plant G-th R q u l . 1962,1,211. 27. Sisler,E.C.;Yang,S.F.Phy&bmistry,19% 23,2765. Overbeek, J. M. M. In Biahamiml and Physlologiml &pet8 o f E t h y b m 28. Veen. H.; Pmduction in Loupr end H;ghar P h f s ; Clijsfers, H.; Depmfi, M.; Mareelle, R.; Poueke M. Van, Eds.; Kluwer Academic Publishers: Bel@um. 1989;109. 29. Thompmn,J. S.; Harlm: K. L.;Whitmy, J . F J A m r Cham Soc 1953,laS.3522. 30. Munskafs, M.; Kitagawa, S. A b s t m ofPapes, Plut 1 International Chemical Congreaa of Pacific Bsaln Societies, Arne-" Chemical Society, 1989. Abstraet N O R 0 151.
