SCIENCE
Mechanism of Ethylene Synthesis In Plants Garified Knowing details of chemical pathway to ethylene could be key to achieving longer plant vitality, increased crop yields, improved harvesting efficiency Ron Dagani, C&EN Washington
In 1893 in the Azores, a small fire broke out in a greenhouse containing pineapple plants. Instead of damaging the plants, the smoke miraculously caused them to burst into bloom. This surprising turn of events is easily explained today: The blaze produced ethylene, a plant hormone that not only promotes flowering but plays a profound role in the growth and development of plants. That plants produce and use their own ethylene was firmly established early in this century. But the key molecular details of how plants synthesize this remarkable two-carbon regulator have started coming to light largely in the past few years. Some of the crucial discoveries are coming from organic chemists who have made inroads into the traditional preserve of plant physiologists and biochemists. Researchers who have been studying the biosynthesis of ethylene for many years still marvel at the compound's diverse effects on plants. Ethylene causes seeds to sprout, flowers to bloom, fruit to ripen and fall off, and leaves and petals to shrivel and turn brown. The agricultural industry uses exogenous ethylene to ripen produce uniformly, making harvesting more efficient. It would be even more efficient, though, to be able to control p l a n t s ' o w n g e n e r a t i o n of ethylene. Likewise, if ethylene bio-
Pirrung: not a concerted mechanism synthesis could be inhibited effectively, one could keep plants vital longer, increasing crop yields. The key to achieving these ends may lie in an intimate understanding of the mechanism of ethylene biosynthesis. Such an understanding received a big boost in 1979 from work done by plant physiologist and biochem-
ist Shang Fa Yang with graduate student Douglas O. Adams at the University of California, Davis. Yang and Adams showed that plants make ethylene from 1-aminocyclopropane1-carboxylic acid (ACC). This unusual amino acid is produced from S-adenosylmethionine, which in turn is the result of hitching an adenosyl group to the common amino acid methionine. With Yang and Adams' discovery, the last blank in the chemical pathway to ethylene was finally filled in. Still open, though, were the mechanistic details of the ACC-toethylene transformation. The Davis researchers initially postulated that oxidase-derived hydrogen peroxide cleaves ACC's cyclopropane ring, releasing a two-carbon fragment as e t h y l e n e . The rest of the ACC molecule, they suggested, ends up as carbon dioxide, formic acid, and ammonia. At the University of Oxford, organic chemist Jack E. Baldwin became interested in the ethylene biosynthesis problem. He and his coworkers used ACC labeled specifically on the unsubstituted two-
Artificial exposure to ethylene ripens crops in field Until scientists discover how to regulate the biosynthesis of ethylene in plants, they must resort to other stratagems to control the physiological processes, such as ripening, that ethylene controls. One commercially proven way is to treat crops with a compound that breaks down into ethylene inside the plant. The best known example of this is Ethrel, Union Carbide's tradename for 2-chloroethylphosphonic acid. This crystalline, water-soluble substance is taken up by plants, where it dissociates via a simple pH-dependent reac-
tion into ethylene, chloride, and inorganic phosphate. Commercially, it has been used to make pineapples, tomatoes, and other produce ripen uniformly so that an entire field can be harvested more efficiently. It also is used to regulate the growth and development of other crops, including wheat, barley, apples, cherries, and, most recently, cotton. A small amount of Ethrel goes a long way because plants are remarkably sensitive to ethylene: They respond to concentrations of less than 0.1 ppmof the gas.
February 13, 1984 C&EN
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Science
Biosynthetic pathway to ethylene is well established
Methionine • Adenosine triphosphate
Adenosine î S-Adenosylmethionine ^CHgS-Adenosine
1 -Aminocyclopropane-1 -carboxylic acid (ACC) I
carbon fragment to prove rigorously that it is indeed the source of the ethylene. They sketched out a possible mechanism involving an enzyme-bound imine intermediate. To Michael C. Pirrung, however, the Yang/Adams mechanism seemed "totally improbable," and the Baldwin hypothesis "not particularly plausible." Pirrung, an assistant professor of organic chemistry at Stanford University, decided to try his hand at the problem. In two recently published papers, he has explored the feasibility of three other possible mechanisms for ethylene biosynthesis. These involve nitrene, nitrenium ion, and radical cation intermediates. The most likely of these possibilities became evident after Baldwin's group and Pirrung independently elucidated the stereochemistry of ethylene biosynthesis. Baldwin and coworkers Robert M. Adlington and Bernard J. Rawlings treated apple slices with cis- or fnws-2,3-dideuterio-ACC. In both cases they observed the production of equal amounts of cis- and fnras-dideuterioethylene [/. Chem. Soc, Chem. Commun., 290, (1983)]. Pirrung fed the cis-labeled ACC to several types of plant tissue and also observed the same lack of stereospecificity in the biosynthesis [/. Am. Chem. Soc, 105, 7207 (1983)]. 22
February 13, 1984 C&EN
"That told us that a concerted mechanism was not operating," Pirrung says, thus working against the intermediacy of a nitrene or a nitrenium ion. Focusing his attention on the remaining possibility—a radical cation intermediate—Pirrung looked to electrochemistry, which he notes is "a very clean way to generate amine radical cations." In his electrochemical system, ris-dideuterioACC is oxidized to dideuterioethylene that is "stereochemically scrambled," just as in plants. This finding, he says, points to a stepwise mechanism for both the electrochemical and biosynthetic reactions. Pirrung postulates that ACC is converted into ethylene through an amine radical cation via two oneelectron oxidation steps. The loss of stereochemistry is due to free rotation of the carbon-carbon bond that evolves into ethylene. What remains nebulous is where the electrons go. The ACC-to-ethylene step is believed to be enzymic, but so far the enzyme has eluded all attempts to isolate and characterize it, although many have tried. The enzyme appears to be associated with the cell membrane. But when the membrane is disrupted to release the enzyme, the enzyme loses its activity. This unfortunate state of affairs has hampered researchers, who still know relatively little about the enzyme. One fact that is known about it is that it requires molecular oxygen to make ethylene. Nevertheless, the exact role of oxygen in the biosynthetic mechanism is a key unresolved issue, according to biochemist Christopher T. Walsh of Massachusetts Institute of Technology. Oxygen could participate via two different routes, one producing hydrogen peroxide, the other, two molecules of water. To distinguish between these two possibilities, Walsh says, one needs to have "some way of quantitatively assessing the fate of the oxygen consumed, and that hasn't been done yet." Despite such uncertainties, Pirrung's model is gaining support because it correctly predicts that cyanide—not formate—is a product of the biosynthesis. Evidence for cya-
nide formation in plant tissue has now been found independently by Yang's group, collaborating with Walsh, and by Pirrung. The cyanide, however, doesn't stick around for very long in the free form because it is rapidly incorporated into asparagine, a fact that biochemists have known for some time. Consequently, when Pirrung treated apple tissue with carbon13-labeled sodium cyanide or ACC similarly labeled on the C-1 of the cyclopropane ring, it's not surprising that he found the label on the 7-carboxyl group of asparagine. With the pathway and most of the mechanistic details of ethylene biosynthesis now established, attention in this area is likely to focus on answering the many remaining questions regarding ethylene biochemistry in plants. For example: What is the nature of the ethyleneforming enzyme? How do plants
Pirrung's mechanism involves radical cations
1 -Aminocyclopropane-1 -carboxylate
Proton abstraction
regulate ethylene biosynthesis? How can the process be inhibited? How Mendeleev's 150th birthday commemorated does ethylene interact with recepSoviet chemists, under the auspices tor sites in plants to exert its diof the Ail-Union Mendeleev Chemical verse effects? Society of the U.S.S.R., are commemResearch aimed at a n s w e r i n g orating this month the 150th anniversathese questions already is well unry of the birth of Russia's most fader way. For example, Pirrung, with mous chemist, Dmitri I. Mendeleev. funds from the Camille and Henry Born in the Siberian town of Tobolsk Dreyfus Foundation, is trying to on Feb. 8, 1834, Mendeleev made his probe the "black box" that is the most important contribution to chemethylene-forming enzyme by observistry—the discovery of the periodicity ing how it processes molecules of of the chemical properties of the ACC carrying different substituents. elements—in 1869, when he was 35 Yang, with support from the Naand a teacher of chemical courses at tional Science Foundation, is looking St. Petersburg University. He spent the for compounds that will inhibit next 20 years developing a periodic ethylene biosynthesis. He also is table of the elements based on his trying to explain how environmendiscovery. He presented his idea of tal stresses, such as lack of water, organizing elements on the basis of trigger e t h y l e n e p r o d u c t i o n in their properties to Western European plants and how this gas helps the scientists in June 1889 in a lecture to searcher in the fields of hydrodynamics, plant cope with the stress. the London Chemical Society, and it meteorology, geology, certain branches As Yang points out, ethylene's has been a cornerstone in teaching of chemical technology (explosives, physiological effects can be blocked chemistry and in predicting the chemipetroleum, and fuels, for example) and either by inhibiting biosynthesis of cal properties of elements ever since. other disciplines adjacent to chemisthe gas or impeding its interaction In Mendeleev's lifetime several new try and physics, a thorough expert of at the plant's target site. The probelements, including gallium, scandium, chemical industry and industry in lem with the latter strategy is that and germanium, were found in nature, general, and an original thinker in the almost nothing is known about the and their chemical behavior exactly field of economy." He was one of the site of ethylene action. Scientists susmatched that predicted by Mendeleev's founders of the Russian Chemical Sopect that the site contains a metal periodic table. ciety in 1869 (now the All-Union ion—possibly copper—and that the Though chiefly remembered in the Mendeleev Chemical Society) and ethylene binds to it. West for his development of the perimade important contributions for his Recently, a group led by bioinodic table, Mendeleev made a number time to the study of solutions. From organic chemist Jeffery S. Thompof other important contributions to Rus1861 to 1890, he taught chemistry at son at Du Pont's experimental stasian chemistry. Russian chemist and St. Petersburg University and at other tion in Wilmington, Del., has been science historian L. A. Tchugayev charinstitutes in that city. From 1892 until studying complexes of ethylene and acterized him as "a chemist of genius, his death in 1907, he was head of the other monoolefins with copper(I). a first-class physicist, a fruitful reCentral Board of Weights & Measures. Their work shows that the coordination chemistry of these complexes "is consistent with the proposed role of copper at the ethylene receptor site of plants" [/. Am. Chem. Soc, 105,3522(1983)]. Researchers at the University of research advances understanding of These experiments do not prove California, Los Angeles, have found how biological systems use metal the presence of copper at the bind- that cupric ions in acetonitrile solu- ions, particularly iron and copper ing site. To do that, Thompson says, tion catalyze the epoxidation of ions, to mediate a variety of reac"someone is going to have to iso- carbon-carbon double bonds—that tions involving oxygen. Valentine, postdoctoral fellow late the binding site itself." This is, the formation of a three-member involves an arduous process of ring containing two carbon atoms Catherine C. Franklin, and gradugrinding up plant tissue, separat- and one oxygen atom [/. Am. Chem. ate students Reuel VanAtta and ing it into fractions, treating these Soc, 106, 814 (1984)]. The research Alice F. Tai studied primarily the fractions with radiolabeled ethylene, was conducted by UCLA chemistry reactions of iodosylbenzene and eiand observing how tightly the gas professor Joan S. Valentine and co- ther trans- or ds-stilbene in an acetois bound. At least two research workers and supported by the Na- nitrile solution of cupric nitrate. In both cases, a major product of the groups, one in North Carolina and tional Science Foundation. The discovery may have commer- reaction was fnms-stilbene oxide. one in Wales, are attempting to isolate the binding site. If they succeed, cial significance because epoxides From analyses of reaction rates and it could serve as a major impetus are important starting materials. products under a variety of condifor plant biochemistry. D More important, Valentine says, the tions, the researchers conclude that
Copper ions catalyze olefin epoxidations
February 13, 1984 C&EN
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&CISnCQ two cupric ions probably complex with the oxygen atom on iodosylbenzene. That complex might react directly with the stilbene. Alternatively, [Cu(III)-0-Cu(III)]4+ might dissociate from the complex and react with the stilbene. Other olefins are epoxidized under the same conditions, Valentine says, and preliminary results suggest that solutions of simple salts of other metals also catalyze the epoxidations. Valentine points out that iron(III)porphyrin complexes also catalyze the epoxidation of olefins. Such reactions have been studied by a number of researchers and are well characterized. The reactions have been used as models to understand the mechanism of oxygen incorporation into organic molecules by biological metalloenzymes containing iron. Porphyrin complexes of chromium or manganese also catalyze oxidations of carbon-carbon double bonds. Metalloenzymes containing copper also are used by biological systems to catalyze reactions involving oxygen, but the mechanisms involved are much less well understood than those of iron enzymes. "To understand how the coppercontaining biological systems work, we decided to look at copper and iodosylbenzene," Valentine says. "We were going to make all sorts of
Valentine: advances understanding of how biological systems use metal ions fancy ligands to put around the metal ion to control the reactivity. Our presumption was that there is something very special about the porphyrin. But we tried the simple copper salt first, and found that it reacted with good yield." The message, Valentine says, "is that the porphyrin is not required for the reaction. The porphyrin may play a role in activating oxygen to get to the metal/oxo species, but our results suggest that it does not confer any special reactivity on that species after it is obtained." D
Monsanto honors acetic acid process developers Three chemists and a chemical engineer are the recipients of Monsanto's Queeny and Thomas/Hochwalt Awards for 1984. Denis Forster received the Thomas/Hochwalt award of a bronze medallion and $25,000 for his research in homogeneous catalysis and carbonylation chemistry. L. Stanley Eubanks, Walter R. Knox, and Frank E. Paulik each received a bronze medallion and shared $50,000 for their efforts in commercializing Monsanto's acetic acid process. The Edgar M. Queeny Award and the Charles A. Thomas and Carroll A. Hochwalt Award were established by Monsanto three years ago. The Queeny Award recognizes technical achievements leading to a ma24
February 13, 1984 C&EN
jor commercial success for the company and the Thomas/Hochwalt Award recognizes fundamental, basic, or applied research that has improved Monsanto's technological leadership and enhanced its reputation in the scientific community. All of the recipients were members of a team associated with the development of Monsanto's acetic acid process, which began in the 1960s. Since its commercial introduction, the process has been used for more than 90% of the new acetic acid capacity worldwide. In citing the contributions of the recipients, Monsanto's senior vice president for research and development, Howard A. Schneiderman, noted that many more than four people made great
contributions to the development of the acetic acid process. Forster was cited specifically for his contributions to the understanding of the catalysis appropriate to the acetic acid process. Most of this work was done in the 1960s and 1970s and is of fundamental importance to the science of catalysis in addition to being of great commercial importance to Monsanto. Schneiderman noted that most of this work was done in a period of about four months and subsequently has been confirmed repeatedly by independent investigators. Paulik and Knox directed the development of the basic chemistry in the company's St. Louis corporate labs before the project was transferred to Texas City, Tex. There Eubanks provided key technical guidance and served as process design supervisor for the first commercial plant, which went on stream in 1970. It is more than serendipitous that the Monsanto acetic acid process was an early example of Q chemistry. The effects of the 1973 Arab oil embargo are frequently cited as the main reason for the new wave of Q syntheses. However, at the awards ceremony in St. Louis late last month, there was little doubt among the assembled chemists and engineers that the virtues of homogeneous catalysis in circumventing severe commercial operating conditions were deserving of as much credit as the embargo. The carbonylation of methanol over iodidepromoted rhodium catalysts to acetic acid must rank as a milestone in the history of successful commercial chemistry. Such a success as the acetic acid process does present the problem of what to do for an encore. The guest speaker, former Bell Telephone Laboratories board chairman William O. Baker, addressed the problem by noting that it is sometimes difficult to achieve a good pace of scientific and technical developments. However, pacing may often be as important as the developments themselves. There seems to be some sort of natural rate at which new developments can be assimilated, and this may be crucial to technical and commercial success. D
