Ethylene: Discovery, Occurrence and Functions

In this article we will discuss about:- 1. Discovery of Ethylene 2. Occurrence of Ethylene 3. Movement 4. Biosynthesis 5. Structure and Activity 6. Regulatory Action 7. Mechanism of Action 8. Functions 9. Tropic Movements 10. Growth 11. Dormancy.

Discovery of Ethylene:

It took several years to establish before it became evident that ethylene (C₂ H₄) was a natural plant hormone in early 1920’s. It was observed that ethylene gas could change the tropic responses of roots, break potato dormancy effectively, induced fruit ripening and leaf abscission. In early 1930’s role of ethylene in inducing flowering in pineapple was brought out.

Gane (1934) established that ethylene was natural product of ripening fruits and possibly stimulated fruit ripening. Subsequently evidences for ethylene production came from flowers, leaves, seeds and even roots. Soon after it came to be effectively realized that ethylene had a profound regulatory activity and could be regarded as a plant hormone. However, the concept received lot of criticism.

Partly because it was an age of auxin discovery and everything was attributed to changes in auxin content either through synergistic interaction or simulation of their effect. For more than a decade ethylene was regarded as an oxogenous chemical which regulated large number of processes in plants.

With the discovery of gas chromatography, it became possible to estimate ethylene levels and soon after in 1969 ethylene came to be accepted as a plant hormone.

Based on the definition by animal physiologists hormone is a substance which is formed in ductless glands and moves in blood stream to perform physiological control of another organ. In plants where there is lack of ductless glands or blood stream the definition has been modified.

Accepting ethylene as a plant hormone needs some amount of stretching of concept since for auxins, gibberellins, etc. one can think them to be translocated via vascular system, but the acceptance of ethylene through gaseous diffusion is a novel variation.

Ethylene is the only gaseous hormone which stimulates transverse or isodiametric growth. Its ability to induce fruit ripening was known for a long time but its recognition as a growth regulator came only recently.

Of all the growth hormones, ethylene has simplest chemical structure (CH₂ = CH₂) and is synthesized from methionine, β-alanine or isoamyl alcohol. It inhibits cell elongation in roots and induces short and wide cells. Several ethylene-releasing substances like ethrel or ethaphon are available.

Occurrence of Ethylene:

Exact chemical techniques to establish the identity and quantitize ethylene from different plant organs had to await the discovery of gas chromatography. However, in a simplified fashion several bioassays can be used to detect the occurrence of ethylene. Earlier workers did recognize that application of auxin stimulated ethylene production in plants.

Likewise during seed germination and fruit ripening there is also ethylene production. It ranges from 0.5 to 5 nl/g^−h. However, in some of the fruits it is as high as 100 nl/g^−h. Application of auxin can heighten ethylene production in the degenerating and fading flowers. There is very high ethylene production which touches a level of 3400 nl/g^−h. Several environmental factors like temperature, oxygen affect the ethylene production.

Under low temperature and low oxygen effect the ethylene production is low and, therefore, storage life of fruits can be improved enormously. Light also affects ethylene production. Plants under stress or injury show high ethylene when a tissue is injured or cut there is very high ethylene production.

Thus, in several succulent fruits, cuts or bruises are avoided to prolong their storage life. In trees, bending of branches also increases the ethylene levels enormously. Tissues exposed to ethylene are stimulated to produce a burst of this hormone. In other words, ethylene production is stimulated by ethylene exposure. The autocatalytic ethylene affects its own production and triggers ripening of fruits.

In Penicillium inorganic phosphate has been shown to regulate ethylene production. There is also an evidence to suggest that in higher plants phosphate plays a role in regulating ethylene biosynthesis. Addition of phosphate to low phosphate cultures inhibited the production of ethylene. It may be added that low phosphate cultures produce high rates of ethylene.

The inhibition was 10-15% in the initial three hours, 50% up to 7 hours and 80% in 10 hours. Coincident with decrease in ethylene productions, ATP content of the cultures also increased. However, mycelium protein did not alter in this period.

In low phosphate medium ethylene production was localized in the fungal cells and not the medium. When pre-climacteric apples were incubated in phosphate buffer it tended to reduce ethylene biosynthesis.

Phosphate buffer also reduced the ethylene production in grape fruit peel slices. Similarly Ca++ ions are also known to affect ethylene production in several systems. These ions stimulate ethylene production individually or in collaboration with low phosphate level. Ethylene production is attributed in microorganisms to the presence of methionine and may partly be due to non-enzymatic reactions.

Apparently inorganic phosphate affects large number of plant processes including metabolism of starch and chloroplast, alkaline ribonuclease, esterase, phosphohydrolases. Phosphate which affects the activity of phosphatases during fruit ripening may increase ethylene production and senescence of fruits, phosphorylation and dephosphorylation of proteins, e.g., ethylene synthesizing enzyme could provide a control of its activity.

Phosphate like Ca++ ions regulates ethylene production through phosphorylation of proteins and regulation of the production of secondary metabolites. One of the suggested hypothesis is that calcium precipitates phosphate in the cells and lowers the inhibitory effect of phosphate on ethylene production. Obviously treatment of fruits with inorganic phosphates should help in suppressing or delaying fruit ripening.

ABA is also reported to increase ethylene production in several plant tissues. It is shown to play a role in ethylene formation, although its mode of action is not known. In wounded citrus tissue GA increased ethylene formation. Similarly when aged albedo tissue is given radioactive L-methionine its carbon-3 is preferentially incorporated into ethylene in comparison with carbon-2 and methylcarbon.

Further L-ethionine and L-calanene inhibited methionine incorporation. Evidences also suggest that ATP may be needed in the conversion of methionine into ethylene. KCN and NaN3 also inhibit ethylene production. The general observation is that the ethylene producing systems are highly labile and their formation may be associated with protein synthesis.

Movement of Ethylene:

Ethylene is a small molecule and moves readily through plant tissues through diffusion processes. It is also soluble in water and lipophilic systems facilitate its movements from one plant organ to another. The movement is usually passive through the plant.

Most recent studies show that ethylene moves like CO₂ and follows Fix law of diffusion. Ethylene movement may also be through air spaces. Presence of thick cuticle precludes ethylene release from the inner tissues.

Biosynthesis of Ethylene:

Methionine is one of the important precursors of ethylene and its effective conversion requires oxygen. Where methionine molecule with carbon atom 3 and 4 were exclusively converted into ethylene molecule the conversion is more rapid in the ripening fruits. When applied exogenously it stimulates physiological processes, like abscission, flower initiation, etc. Specific pH is needed for its conversion to ethylene.

Fatty acids are another substrate for ethylene biosynthesis. Accordingly linolenic acid may serve as an ethylene precursor. Similarly lipoxidase is also correlated with ethylene formation in apples. Likewise its activity may control not only metabolism of fatty acids but also peroxides utilization. Emasekl and Watanabe (1978) have reported a reduction in ethylene production due to osmotic shock.

When osmotic shock was applied to IAA pre-treated tissue there was also inhibition of ethylene production. Apparently the activity of induced ethylene producing system was affected by this treatment. The mechanism of the inhibitory effect of the continuous presence of mannitol was different from that of osmotic shock. Osmotic shock possibly modified the membrane structure and inhibited ethylene production.

These authors have suggested that osmotic shock treatment released membrane proteins which were essential for membrane function. In brief, ethylene production was controlled by membrane activity.

Similarly β-alanine and methionine are reported to increase ethylene biosynthesis. Several non- enzymatic systems are used to study ethylene formation. It is generally suggested that methionine is converted into ethylene in the presence of light and FMN. It is suggested that methionol serves as an intermediate when methionine is converted into ethylene.

Yet another intermediate is α-keto-ϒ- mercaptobutarate. There is lot of controversy regarding cellular location of ethylene biosynthesis. Tissues rich in mitochondria produce more ethylene, but the synthesizing system appears to be in the cytoplasm.

In addition to the above mentioned precursors glucose, fumarate, propanol and pyruvate are also regarded as probable precursors of ethylene. Most of these precursors require peroxidase for their conversion.

The regulatory control of ethylene biosynthesis may vary in different tissues under different physiological conditions. Wide range of chemicals are known to stimulate ethylene production and auxin is one of them. Several growth regulators which stimulate growth can stimulate ethylene formation. Ethylene formation can be stimulated by iodoacetate, ascorbic acid, cyclohexamide, etc.

Likewise—hydroxyethylhydrazine, chloroethyl phosphonic acid, aluminium, cobalt are prominent compounds which stimulate ethylene production. In recent years several chemicals have come to fore which suppress ethylene biosynthesis and silver nitrate is one of them.

Figure 20-13 shows three step pathway for ethylene biosynthesis in higher plants. In the first step, an adenosine group is donated to methionine by a molecule of ATP, thus forming SAM. Conversion of methionine to SAM is catalyzed by the enzyme methionine adenosyl transferase.

The cleavage of SAM yields 5′-methylthio-adenosine (MTA) and ACC, mediated by ACC- synthase. This is a rate limiting step. ACC synthase is the only enzyme in the pathway that has been studied in details. In tomato this enzyme has been partially purified, is present in small quantity and is highly instable.

Recently genes for ACC synthase have been isolated from tomato pericarp tissue. The cloned genes direct the synthesis of active ACC synthase in E. coli and yeast. Conversion of ACC to ethylene is mediated by ethylene forming enzyme (EFE). However, it may be stated that ethylene biosynthesis is limited due to small amount of free methionine available in plants.

Structure and Activity of Ethylene:

Ethylene-like activity seems to be an unsaturated bond next to a terminal carbon. The effectiveness of CO in ethylene system was noted long ago. It is usually suggested that ethylene binds to a metal containing receptor sites as silver and mercury. In fact ethylene is a most prevalent unsaturated hydrocarbon produced by the plants.

Regulatory Action of Ethylene:

Ethylene appears to regulate wide spectrum of physiological processes. It seems to inhibit growth especially along its length. The lateral enlargement of roots may be due to ethylene stimulation of lateral growth. It also appears to be associated with geotropic bending of roots, suppression of bud growth, etc.

Mechanism of Action of Ethylene:

In general, three theoretical lines of ethylene action have been suggested; it becomes attached to some metalloprotein site in the cell which possibly acts as a regulator; it is attached to membrane layer and it may regulate RNA and RNA-directed protein synthesis. Burg and Burg (1977) proposed metal-adsorption theory which was based on two evidences.

First, changes in biological activity with molecular structure of different substances like acetylene. CO, 1-butene, etc. were similar to the alteration in ethylene adsorption on to a heavy metal like silver. Second, CO₂ inhibits ethylene attachment to a heavy metal.

In fact, interference of CO₂ with ethylene responses has been shown for many ethylene systems including abscission, stimulation of epicotyl hook formation in etiolated seedlings, root growth inhibition and stimulation of fruit ripening.

One of the standard tests for ethylene stimulated processes is their reversal by CO₂. The interaction between ethylene and O₂ has also been demonstrated. In fact, depressed levels of oxygen inhibit ethylene action. One of the views is that oxygen was essential for oxidation of the metal receptor site where ethylene was attached. Indeed, storage of fruits could be prolonged in controlled conditions with low oxygen and high CO₂ levels.

Some of the workers have used deutrated ethylene and studied the nature of its binding site. Here the plants were exposed to such an ethylene and deuterion displacement by hydrogen atoms, etc. was examined.

In these experiments ethylene recovered was unchanged indicating that attachment was neither by covalent nor by hydrogen bonding. Zinc is the additional metal site suggested for ethylene attachment and carbonic anhydrase adsorbed it.

According to the second theory ethylene becomes attached to membrane surface, possibly the lipid layer. When supplied, ethylene changes the permeability of mitochondrial membrane. However, some workers have contradicted these observations and suggested that ethylene had very little effect on surface tension of the membrane.

Yet additional mechanism of ethylene action appears to be the possible changes of enzyme patterns by affecting RNA directed protein synthesis. Ethylene alters several enzymes including peroxidase, phenolases, etc. Enzymes concerned with phenolic metabolism are also changed by ethylene.

Ethylene is known to increase phosphatases, esterases as well as cellulase. The stimulation of abscission by ethylene seems to be associated with an increase in some cellulases through de novo synthesis. Ethylene also seems to stimulate release of bound cellulase into the free form. Apparently ethylene may induce enzyme changes not essentially by altering their biosynthesis.

Several of ethylene effects have been retarded by inhibitors of protein and nucleic acid synthesis which implies that this hormone regulates enzyme synthesis through RNA directed synthesis. It may be added that ethylene may be regulating growth functions in a highly complex fashion. Further ethylene has also been shown to affect polarity of growth, thus changing the tropic responses.

In fact, stimulation of lateral cell enlargement is mediated by ethylene and the process may be affected by changes in polar auxin transport. Ethylene treatments have been shown to alter mitotic activity and arrangement of cellulose microfibrils in the cell walls. This may be due to the disorientation of microtubles which may be concerned with mitotic spindle mechanisms.

Functions of Ethylene:

Abscission:

Ethylene stimulates abscission of leaves, fruits and flowers. It has been shown by Rasmusson and his associates that the action of ethylene is mediated through the enhancement of acid phosphatase and other hydrolases.

Senescence:

Ethylene induces yellowing of leaves and downward bending. The flowers may even fade in its presence and wither. Presence of ethylene induces colour changes in flowers.

Tropic Movements of Ethylene:

Ethylene makes the stems ageotropic possibly by inhibiting polar transport of auxin. However, ethylene is essentially required for geotropic response of stem.

Growth of Ethylene:

In general ethylene has inhibitory effect and retards longitudinal growth. On the other hand it promotes isodiametric and transverse growth. It promotes pollen tube growth.

Dormancy of Ethylene:

Ethylene can break dormancy of several plant organs but inhibits lateral bud growth.

Rooting:

In Phaseolns, rooting of cuttings is enhanced through ethylene application.

Flowering:

Ethylene induces flowering in pineapple.

Fruit Ripening:

The primary role of ethylene seems to be in fruit ripening. The process is well marked in climacteric fruits. The fruit dehiscence may also be affected by ethylene. It also affects peroxidase levels.

The role of ethylene in affecting polar transport of auxin has also been proposed by some workers. The role of ethylene producing compounds on sex reversion and formation of female flowers has also been demonstrated in Cannabis and papaya.

Carbon dioxide competes with ethylene actions in several responses. They may react at common binding sites. The inhibition may be through some enzyme reaction. We do not know the primary reaction site for ethylene. It is known to enhance amylase activity. It may affect membrane permeability.