FEATURE ESSAY 11.1 A century of ethylene research

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Barry McGlasson

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Figure 1 Barry McGlasson, University of Western Sydney Hawkesbury, Richmond Campus, at his gas chromatograph (fitted with flame ionisation detector) analysing ethylene concentration in gas samples taken from air exiting enclosed containers of harvested plums.

  Plants, fungi and bacteria produce a host of volatile com-pounds. Some attract or repel animals, some create powerful emotions in humans and some induce morphological and metabolic changes in adjacent plant tissues. Of all these emanations only ethylene is recognised as a natural gaseous plant hormone.

Ethylene has been used unintentionally to manipulate crops such as fig as far back as the third century bc. The sycamore fig originated in eastern central Africa, where it was naturally pollinated by a small wasp that makes its home inside the fruit. When the sycamore fig was taken into the eastern Mediterranean countries, including Egypt, pollinating wasps were left behind. Nevertheless, young fruit which were mechanically injured set parthenocarpically and ripened without seed! A 1633 herbal noted that ‘It bringeth forth fruit oftener if it be scraped with an iron knife, or other like instrument’. The fruit is ‘like in juice and taste to the wilde fig, but sweeter, and without any grains or seeds within’. We now know that wounding young fruit would have stimulated ethylene production and this gas induced those figs to grow and develop parthenocarpically. David Blanpied summed up this piece of history by recasting Amos 7:14 (OT), ‘I was no prophet, neither was I a prophet’s son; but I was an herdsman, a gatherer of sycamore fruit’, as ‘I was an herdsman and an activator of ACC synthase in sycamore figs’ (Blanpied 1985).

Blanpied’s quotation nicely sums up the history of ethylene as a plant hormone because it takes us from simple fruit behaviour to underlying biochemistry. Once the presence of ethylene in plant emanations was proved chemically, a lively debate followed as to whether a gas could really be defined as a hormone. There were two major developments that resolved this issue. First was the invention of gas chromatography which soon enabled measurement of ethylene at concentrations that were physiologically meaningful and in small gas volumes (Burg and Stolwijk 1959). Second, a non-volatile plant product, 1-aminocyclopropane-1-carboxylate, was found to be the immediate precursor of ethylene (Adams and Yang 1979). Any lingering doubts that ethylene was a plant hormone have now been completely erased by application of molecular methods.

This story of ethylene mixes applications of plant physiology with human intuition, and is conveniently related to three eras that represent technical evolution in this area of plant science, namely, pre-1935 (an age of mystery), 1935–1979 (an age of enlightenment) and post-1979 (an age of opportunity).

An age of mystery

In 1858, Fahnestock in the USA observed that illuminating gas caused plant senescence and leaf abscission, and Girardin (1864) in France subsequently showed that ethylene was a component of illuminating gas. Many suspected that such plant responses were due to ethylene, but it took a Russian student, Neljubov (1879–1926), to establish that ethylene is a biologically active compound. As a young man he observed that pea seedlings germinated in the dark grew in a horizontal direction when exposed to laboratory air containing burnt gas. He showed that the plants resumed normal growth when the air was first passed over heated CuO to oxidise hydrocarbon gases. This growth response was used as a bioassay for the next 50 years. We now know that these pea seedling responses are induced by as little as 0.06 µL L–1 ethylene.

Many publications from around 1910 indicated that ethylene was produced by ripening fruit such as pears and apples. By 1923, Denny (US Department of Agriculture) had patented ethylene for ripening bananas, tomatoes and pears, removing astringency from persimmons and loosening walnut husks. Finally (1934) Gane in Britain produced conclusive proof that ethylene is a natural product of plants, and to obtain enough ethylene for his tests he collected gases from about 28 kg of apples. He extended this proof to other fruits a year later.

An age of enlightenment

Following Gane’s confirmation that ethylene generation was common in fruits, research interests broadened beyond this simple ethylene–fruit connection. By 1940 the postharvest pioneer Jacob Biale (University of California, Los Angeles) showed that green citrus mould (Penicillium digitatum) also produced ethylene, thereby extending ethylene physiology to plant–fungus interactions. Hormonal interrelations entered this picture when the synthetic auxin 2,4-D was later shown to stimulate ethylene production by plants (Morgan and Hall 1962), confirming earlier indirect observations on auxin responses (Zimmerman and Wilcoxon 1935). Ethylene was by now acknowledged as instrumental in fruit ripening, but a nagging question remained as to whether ethylene was a true ripening hormone or merely a by-product of ripening events (Biale et al. 1954). I entered the field at this stage, and to resolve this issue of hormone status we needed to establish whether ethylene production by fruits increased ahead of ripening. Progress in unravelling cause and effect would hinge on development of a sensitive assay for ethylene.

Strong indications of ethylene involvement in ripening came from Workman and Pratt (1957). These authors used cold mercuric perchlorate solutions to bind specifically ethylene rather than other gases, and thus trap a sufficient amount from ripening tomatoes to measure it manometrically. However, the much greater sensitivity of gas chromatography subsequently allowed Burg and Stolwijk (1959) to demonstrate via frequent monitoring that ethylene production actually precedes the onset of ripening in some fruit.

Scientifically, these were exciting times. As a PhD student at the University of California, Davis, I was a member of one of the first teams to use a gas chromatograph fitted with a flame ionisation detector to measure internal ethylene con-cen-trations in a ripening fruit (Lyons et al. 1962). We showed conclusively that cantaloupe (rockmelon) was climacteric. Harvested fruit showed an increase in ethylene production with onset of a respiratory climacteric and ripening.

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Figure 2 Ethylene generation influences postharvest behaviour of Cymbidium flowers. When the pollen cap is removed from the floral column either by insect pollination or by human mishandling, endogenous ethylene production is triggered in that flower (left side), bringing about anthocyanin synthesis, cupping of petals and swelling of column tissues within 3 d. Intact flowers (right side) remain fresh for three weeks. Scale bar = 1 cm.

(Photograph courtesy R.L. Bieleski)

Over the next 20 years an explosion of publications docu-mented ethylene involvement in many plant responses. Burg and Burg (1960s) demonstrated that ethylene was essential for ripening as well as other developmental events in plants. Senescence is a case in point, and a clear ethylene response is shown in Figure 2 for Cymbidium flowers.

A further practical development from ethylene research dates from 1963 with synthesis of ‘Ethephon’ (2-chloroethyl-phosphonic acid) (also called ‘Ethrel’). This water-soluble compound is readily absorbed by plants, and breaks down to release ethylene above pH 4.6. Ethephon thus provides a convenient way of applying ethylene to plants under field con-ditions and is still widely used to promote uniform maturation of processing tomatoes as an aid to mechanical harvesting.

Three broad research themes in ethylene physiology were now underway: mode of action, inhibition of action and biosynthesis. However, a major problem confounding our best efforts in all three areas was the autocatalytic behaviour of ethylene. This gas stimulates its own production, so how do you distinguish between the external ethylene you have applied experimentally as a stimulus, and the endogenous ethylene which is produced as a response by the plant tissues? Con-fronted by this dilemma, we devised a neat trick based upon a closely related gas (McMurchie et al. 1972). Propylene is a three-carbon analogue of two-carbon ethylene, and can stimulate typical ethylene responses! Moreover, propylene is also easily distinguished from ethylene by gas chromato-graphy. We now had an elegant tool for analysis of ethylene physiology.

Spurred by this development, we applied propylene to citrus fruit (non-climacteric) and to bananas (climacteric) to mimic an exogenous ethylene stimulus, and measured endo-genous ethylene production directly. Citrus respiration was stimulated without any increase in ethylene production, whereas in banana both respiration and endogenous ethylene production were stimulated. These outcomes were consistent with our paradigm of ripening in climacteric versus non-climacteric fruit.

Once ethylene was widely acknowledged as a ripening hormone, there was a strong demand by industry for practical control methods in order to extend fruit storage life. Our original approach was to remove ethylene from fruit storage atmospheres by scrubbing with oxidising agents such as per-manganate. Commercial absorbents containing permanganate are available but inconvenient to use because the absorbent has to be packaged to prevent contact with stored fruit. The search for other ways of avoiding or inhibiting ethylene action continued. By 1976, Beyer showed that silver ions are a potent inhibitor of ethylene action, and a new set of management options opened up immediately. Ag+ is readily bound by plant tissue but not easily translocated and thus of limited ap-plication. However, the silver thiosulphate complex (STS) is negatively charged and can move readily through plant tissues. This observation had little practical value for fruits which are eaten, but has had wide use in slowing the ethylene-driven senescence of cut flowers. In 1979 Sisler introduced volatile unsaturated ring compounds as inhibitors of ethylene action, the most potent being norbornadiene. Sisler has subsequently developed 1-methylcyclopropene (1-MCP), a gaseous com-pound which is essentially an irreversible inhibitor and safe to use (Sisler and Serek 1997).

While ethylene was gaining wider application in post-harvest physiology, research continued with unravelling the biosynthetic pathway. The first clue came when Lieberman and Mapson (1964) supplied the general precursor 14C-methionine to ripening tissue and found 14C activity in the ethylene produced. Methionine had been noted as a possible precursor from the discovery that rhizobitoxin inhibits ethylene production (Owens et al. 1971). Rhizobitoxin inhibits pyridoxal phosphate-containing enzymes of the kind involved in methionine-utilising pathways. Adams and Yang (1977) working at UC Davis then showed convincingly that S-adenosylmethionine (SAM) rather than methionine was a key precursor in ethylene biosynthesis. Two years later they topped this triumph by discovering the immediate precursor of ethylene, namely 1-aminocyclopropane-1-carboxylate (commonly abbreviated to ACC). Within another few years, Yang and co-workers had managed to define the biochemical pathways that generate ACC from methionine via SAM.

An age of opportunity

Major advances in our knowledge of ethylene biosynthesis and physiological roles have come from gene technology. The chain of biochemical steps from SAM to ethylene is now known, and the genetic codes for enzymes involved have been defined. Detailed characterisation of ACC oxidase in particular was a significant outcome of gene technology. Despite years of effort, all attempts to extract the enzyme had failed. It was widely believed to be associated with the cell membranes, because activity was lost once the cells were disrupted. Recognising that each protein (enzyme) is linked to its unique gene (DNA) through a unique mRNA, Grierson and co-workers searched for mRNAs that increase during tomato ripening. This was a predicted behaviour for the mRNA modulating ACC oxidase, an enzyme that increases markedly in tissue activity during ripening. Expression of one particular cDNA clone enabled the enzyme to be ‘fished’ from tissue extracts. As a result this enzyme proved to be soluble (cytoplasmic) and not membrane bound.

Increased awareness of genes involved in ethylene biology creates opportunities for answering many remaining questions about ethylene-driven behaviour. One that continues to hold my curiosity concerns regulation of ethylene production in relation to ontogeny of plant organs, an issue unresolved since my early work with developing tomato fruit. In those experiments, I observed that tomato fruit harvested less than 15 d after anthesis failed to undergo normal ripening whereas fruit harvested somewhat later began ripening in a few days (McGlasson and Adato 1977). How then are ethylene-driven events coordinated with organ ontogeny?

Answers will come when we understand the mode of ethylene action, and as an initial step analogous to other areas of hormone physiology ethylene receptors have now been identified (Section 9.2.1).

Molecular tools offer new clues, given the exciting dis-covery of mutant genes from Arabidopsis thaliana plants that are insensitive to ethylene. Insensitive mutants have enabled isolation of the ETR1 gene which encodes a protein that binds ethylene and is antagonised by competitors of ethylene action. Similarly, a ripening-impaired mutant tomato (Never Ripe) has been found to contain a homologue of ETR1 that encodes proteins lacking the ability to receive ethylene (Section 9.3.3). The question remains of how many kinds of ethylene receptors there are.

In 1972 we established a distinction between climacteric and non-climacteric fruit in their response to exogenous ethylene that led us to propose two systems for regulation of ethylene production. System 1 would be responsible for back-ground ethylene production found in non-climacteric fruit and in pre-climacteric fruit. System 2 would account for the increased ethylene production associated with ripening in climacteric fruit. It will be especially gratifying if future devel-opments confirm our original model.

As a step in this direction, we know that propylene induces a rapid increase in respiration without any increase in endo-genous ethylene production with pre-climacteric fruit. 1-MCP has turned out to be a very useful tool for distinguishing between events regulated by ethylene and those that are independent. We have found that 1-MCP prevents the initial increase in respiration in Japanese plums induced by propylene as well as delaying normal ripening and the accompanying ethylene and respiratory climacterics. Because 1-MCP binds so strongly it could greatly assist our efforts to recover and characterise native ethylene receptors.

Gene technology in conjunction with better recognition and utilisation of natural fruit variants will almost certainly enable us to develop cultivars of highly perishable commodities such as stonefruit that will cool store longer and have better shelf life. Transgenic tomatoes and melons already exist which require ethylene treatment to ripen. Natural mutants of nectarines have been reported that require ethylene treatment to ripen and we have described two cultivars of slow-ripening Japanese plums with a suppressed ethylene climacteric pheno-type (Abdi et al. 1997). In contrast to normal cultivars, when treated with 1-MCP the slow-ripening plums seem unable to regenerate ethylene receptors unless they are treated with propylene. (This application calls for propylene so that pro-duction of endogenous ethylene can be detected.)

Once applied, these new technologies could provide horti-culture with ‘designer fruit’ which are initially insensitive to ethylene and consequently easy to store. As required, this stored fruit could then be dosed with gas to produce ethylene receptors and thus trigger ripening. As shown experimentally, propylene would work, but ethylene will be preferred for commercial applications on grounds of lower cost and higher activity.

References

Abdi, N., Holford, P., McGlasson, W.B. and Mizrahi, Y. (1997). ‘Ripening behaviour and responses to propylene in four Japanese type plums’, Postharvest Biology & Technology,
12, 21–34.

Adams, D.O. and Yang, S.F. (1977). ‘Methionine metabolism in apple tissue’, Plant Physiology, 60, 892–896.

Adams, D.O. and Yang, S.F. (1979). ‘Ethylene synthesis: identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene’, Proceedings of the National Academy of Science, USA, 76, 170–174.

Biale, J.B., Young, R.E. and Olmstead, A.J. (1954). ‘Fruit respiration and ethylene production’, Plant Physiology, 29, 168–174.

Blanpied, G.D. (1985). ‘Introduction to the symposium, Ethylene in postharvest biology and technology of horticultural crops’, HortScience, 20, 40–41.

Burg, S.P. and Stolwijk, J.A.A. (1959). ‘A highly sensitive katharometer and its application to the measurement of ethylene and other gases of biological importance’, Journal of Microbiological and Technological Engineering, 1, 245–259.

Lieberman, M. and Mapson, L.W. (1964). ‘Genesis and biogenesis of ethylene’, Nature, 204, 343–345.

Lyons, J.M., McGlasson, W.B. and Pratt, H.K. (1962). ‘Ethylene production, respiration, and internal gas concentrations in cantaloupe fruits at various stages of maturity’, Plant Physiology, 37, 31–36.

McGlasson, W.B. and Adato, I. (1977). ‘Relationship between the capacity to ripen and ontogeny in tomato fruits’, Australian Journal of Plant Physiology, 4, 451–458.

McMurchie, E.J., McGlasson, W.B. and Eaks, I.L. (1972). ‘Treatment of fruits with propylene gives information about the biogenesis of ethylene’, Nature, 237, 235–236.

Morgan, P.W. and Hall, W.C. (1962). ‘Effect of 2,4-diclorophenoxyacetic acid on the production of ethylene by cotton and grain sorghum’, Physiologia Plantarum, 15, 420–427.

Owens, L.D., Lieberman, M. and Kunishi, A. (1971). ‘Inhibition of ethylene production by rhizobitoxine’, Plant Physiology,
48, 1–4.

Sisler, E.C. and Serek, M. (1997). ‘Inhibitors of ethylene responses in plants at the receptor level: recent developments’, Physiologia Plantarum, 100, 577–582.

Workman, M. and Pratt, H.K. (1957). ‘Studies on the physiology of tomato fruits. II. Ethylene production at 20°C as related to respiration, ripening and date of harvest’, Plant Physiology,
32, 330–334.

Zimmerman, R.H. and Wilcoxon, F. (1935). ‘Several chemical growth substances which cause initiation of roots and other responses in plants’, Contributions Boyce Thompson Institute of Plant Research, 7, 209–229.

Further reading

Abeles, F.B., Morgan, P.W. and Saltveit, M.E. (1992). Ethylene in Plant Biology, 2nd edn, Academic Press: San Diego.

Wills, R., McGlasson, B., Graham, D. and Joyce, D. (1998). Postharvest: An Introduction to the Physiology and Handling of Fruit, Vegetables and Ornamentals, 4th edn, UNSW Press: Sydney.

 

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