In this article we will discuss about Ethylene. After reading this article you will learn about: 1. Biosynthesis of Ethylene 2. Responses of Plants to Ethylene 3. Mode of Action.
Biosynthesis of Ethylene:
Lieberman and Mapson (1964), first proposed that the amino acid methionine is the precursor of ethylene. Adams and Yang (1979) established the sequence for the pathway for ethylene biosynthesis in ripening apples which is like this — Methionine — SAM (S-adenosylmethionine) — ACC (1 -aminocyclopropane-1 -carboxylic acid) — ethylene.
First methionine is converted to S- adenosylmethionine (SAM) by reacting with ATP. The next step in the pathway is the conversion of SAM to ACC and MTA (methylthioadenosine). ACC synthase, which catalyses the conversion of SAM to ACC and MTA plays a key role in regulating ethylene biosynthesis.
In this process, the CH3S group of methionine is released from SAM and MTA which is then rapidly hydrolyzed to methylthioribose (MTR) and the CH3S group of MTR is recycled back into methionine. More recently, Yung et al. and Wang et al. showed that the ribose moiety of MTR is directly incorporated into 2- amino butyrate moiety of methionine.
Regarding the source of 2-amino butyrate, Adams and Yang (1977) proposed that MTR donates its CH3S group to a 4-carbon acceptor probably homoserine or its related analogue to form methionine, while the ribose moiety of MTR is split off. Another proposal is that the ribose unit of MTA or MTR directly provides the carbon skeleton of methionine along with CH3S group.
In view of the limited amount of methionine present in plants, sulphur of methionine in the form of CH3S group must be recycled back to methionine, whereas 3,4-carbon moieties which contribute to ethylene production are ultimately replenished from the ribose portion of ATP.
The last step in the ethylene biosynthesis, the conversion of ACC to ethylene, is carried out by an oxidative enzyme ACC oxidase which was previously called the “ethylene-forming enzyme” (EFE).
It has been shown that the intermediates including ACC are formed under anaerobic condition but its conversion to ethylene depends on the presence of oxygen. ACC synthase is a cytosolic enzyme and its activity is regulated by several environmental factors like drought stress, wounding, flooding and internal factor such as the level of auxin.
The enzyme ACC synthase is inhibited by aminoethoxyvinylglycine (AVG) and aminooxyacetic acid (AOA) and thereby ethylene production may be blocked. The cobalt ion (Co2+) is also an inhibitor of ethylene formation, which blocks the conversion of ACC to ethylene, the last step in ethylene biosynthesis.
Hoffman et al. noted that in addition to its conversion to ethylene, ACC can be metabolized to N-malonyl ACC (MACC). The enzyme catalysing this conversion is ACC N-malonyl transferase which is present in a wide range of plant tissues.
Regarding the physiological role of MACC, it was initially proposed that malonylation of ACC serves as a means for storing ACC as an inactive product to prevent overproduction of ethylene that could be hydrolysed back to ACC when ethylene production is needed.
Now it is thought that MACC does not act as a source of ACC, instead it is a sink that represents a detoxified end product of ACC and thus reduces ethylene production.
When leaves are subject to water stress, ethylene production is increased which is caused primarily by an increase in ACC synthesis. All the plant hormones including ethylene regulate ethylene production at the level of ethylene synthesis.
Auxin promotes ethylene production by inducing the production of ACC synthase, resulting in an increased level of ACC, eventually leading to an increase in ethylene production. ABA effectively reduces ethylene production in leaves specially under wilting condition or when ethylene production is stimulated by IAA. Cytokinins, on the other hand, stimulate ethylene production.
The pro-motive effect of IAA and water stress on ethylene production is further enhanced by cytokinin. Ethylene can promote its own synthesis in intact ripening fruits—this phenomenon is called autocatalysis. In excised fruit tissues, ethylene production can be inhibited by ethylene treatment and this is known as auto inhibition.
Responses of Plants to Ethylene:
(i) Fruit Ripening:
On the basis of the observation that ethylene can stimulate fruit ripening, it has gained recognition as a ripening hormone. The stimulation of ripening by ethylene seems to be restricted to climacteric fruits like bananas, tomatoes, melons and avocados in which ripening is associated with a sudden increase in respiration or ethylene production.
In such fruits, a relationship between ethylene production and respiration has been established. During climacteric rise in respiration, there is a massive increase in CO2 release followed by a decrease. It is also to be noted that a climacteric rise in ethylene production precedes the climacteric rise in CO2 production, suggesting that ethylene is the hormone that triggers the ripening process.
Ethylene appears to have no role in non- climacteric fruits like oranges, lemons and grapes which do not show sudden increase in respiration during ripening.
(ii) Release of Dormancy:
In a number of species ethylene application stimulates the germination of dormant seeds and thus may prolong seed longevity. Normal dormancy is probably related to less ethylene evolution. It has been shown that treatments that break dormancy increase ethylene evolution.
(iii) Growth and Differentiation of Shoot and Root:
Ethylene produces spectacular effects on the growth and development of etiolated seedlings. The physiological action of ethylene causes the so-called triple response which involves a reduction in elongation, swelling of the hypocotyl and a change in the direction of growth.
There is an increase in stem diameter which indicates that lateral growth as opposed to longitudinal growth is favoured by ethylene. It has been suggested that ethylene redirects the orientation of new cell-wall micro fibrils from longitudinal to radial direction.
Ethylene exposure in plants causes downward growth of the petioles, termed epinasty which seems to result from a redistribution of auxin in response to ethylene treatment. Increased growth in the upper part of the petioles causes increased growth in that region resulting in a downward bending of the petiole.
Root growth is stimulated at low concentrations of ethylene and inhibited at higher concentrations. Root anatomy can also be affected by the endogenous ethylene content.
The development of aerenchymatous roots in flooded maize plants is an example of this response. This adaptive response to increased intracellular ethylene can also be induced by exogenous ethylene application. Another interesting response of root to ethylene is the enormous production of root hairs.
(iv) Responses to Physical Stimuli:
While the ability of plants to respond to a number of physical stimuli like gravity and light has been correlated with changes in the distribution of auxin, ethylene has been shown to be an active agent in some cases.
The response of some plants to tactile stimuli appears to be mediated through an increased production of ethylene. Root hairs are formed in many climbing vines to attach the plants to their support. In this case, ethylene formed as a result of gentle stimulation like localized contact is thought to stimulate root hair development and ethylene may be the signal to improve the support.
(v) Adventitious Root Formation:
Ethylene stimulates the formation of adventitious roots, leaves, stem and pre-existing roots. It has been demonstrated that high concentrations of auxin produce ethylene and auxin-induced rooting of cuttings appears to be due to increased ethylene production.
Ethylene accelerates the abscission of plant organs. Research on the hormonal control of abscission has revealed that a gradient of auxin must be maintained from the leaf or fruit to the plant axis in order to delay or reduce abscission. This gradient is maintained by juvenility factors like auxin, cytokinin, light and good nutrition.
When such auxin gradient is disturbed or reversed, the abscission zone becomes sensitive to ethylene.
Once sensitized, the cells of the abscission zone respond to low concentration of ethylene by the production of cell-wall hydrolysing enzymes followed by the shedding of the organ. When ABA causes abscission, it may do so either by stimulating ethylene formation or by interfering with auxin synthesis or its transport from the leaf.
(vii) Flower Induction and Opening:
One of the commercially important effects of ethylene is the induction of flowering in pineapple, mango and apple. The opening of flowers may be effected in different ways by ethylene. For example, the opening of carnation buds is accelerated by ethylene, whereas it inhibits the opening of rose buds at similar concentrations. The mechanism by which ethylene modifies the flowering process is not clear.
(viii) Flower and Leaf Senescence:
In many flowers, senescence is associated with a considerable amount of ethylene production. This intracellular ethylene-induced flower senescence can also be induced by treatment with exogenous ethylene or ACC and prevented by inhibitors of ethylene synthesis or action.
In 1984, Reid and his associates used Petunia hydrida L. as an ideal experimental system for studying flower senescence wherein the colour changes from pink to purplish-blue during senescence. Like climacteric fruits, senescence of aged flowers can be caused by less ethylene because they become increasingly sensitive to ethylene as they age.
Studies by Reid and others (1983) on the control of flower senescence by ethylene have revealed that pollination causes a very rapid increase in ethylene production first by the gynoecium, and then the petals. Since a rapid senescence of some flowers takes place immediately after pollination, it has been suggested that pollination acts as a stimulus the nature of which is like ethylene or ACC, the precursor of ethylene.
(x) Wound Responses:
When plants are wounded or exposed to stress conditions, ethylene production rapidly rises. In such cases, ethylene acts as a wound hormone which seems to reduce stress or to withstand infection.
Ethylene can also lead to the production of phytoalexins in wounded plants which are compounds meant to overcome fungal infection. A number of secretory processes like gum production and latex flow are stimulated by ethylene.
Mode of Action in Ethylene:
The mode of action of ethylene is not well understood. We now know much about the biochemical changes associated with ethylene-mediated plant responses. On the contrary, very little is known of the mechanism by which ethylene mediates such processes. It remains to be elucidated how the simple hydrocarbon ethylene has such spectacular effects on plant development.
(i) Ethylene Receptor Involved in Signaling and Site of Action:
Ethylene has been shown to induce specific changes in genetic expression.
By analogy to other plant hormones, molecular biological phenomena like the synthesis of new mRNA and protein can be controlled by ethylene. It is thought that like other plant and animal hormones, ethylene probably mediates its action through a receptor which is protein in nature and all of these changes must somehow be mediated by the ethylene- binding protein.
Burg and Burg proposed that the ethylene receptor site contains a metal, which is either zinc or copper. Ethylene is thought to bind to a receptor molecule that is activated as a result of this interaction. The activated receptor molecule triggers the primary response which then initiates a chain of reactions leading to the physiological response.
In several instances, ethylene metabolism appears to be connected with ethylene action. It has been noted that in plant tissues, ethylene is oxidized to CO2, ethylene oxide and ethylene glycol. There is a positive correlation between the metabolism of ethylene and the responsiveness of the tissue to ethylene.
Beyer has proposed the hypothesis that ethylene action and metabolism might be coupled and according to this hypothesis, the response to ethylene is initiated through the interaction of ethylene with a metal containing receptor and the same metal-containing site is thought to catalyse the oxidation of ethylene.
Oxidation of Ethylene:
Ethylene can be oxidized to ethylene oxide. Subsequently, ethylene can be hydrolysed to ethylene glycol.
In most plant tissues, however, ethylene has been found to be completely oxidized to CO2.
Our knowledge about ethylene receptor is largely extended by molecular genetic studies of Arabidopsis thaliana, whereby different classes of mutants have been isolated differing in their sensitivity to ethylene.
The ethylene receptor protein is encoded by the gene ETR 1 (ethylene resistant 1). ETR 1 is a trans membrane protein which shows sequence similarity to bacterial two-component histidine kinases.
The two components consist of:
(i) A sensor histidine kinase by which bacteria sense and respond to different environmental factors and
(ii) A response regulator.
A model has been proposed which gives an idea about how the ethylene signal is perceived on the membrane and transduced along various protein components ultimately resulting in gene expression and physiological action. Although the exact location of all these proteins is not yet understood, it is possible that ETR 1 receptor with which ethylene binds is a membrane-bound protein.
This is followed by inactivation of the CTR1 (constitutive triple response 1) protein which acts as a negative regulator of the signaling pathway.
Other proteins, which become active in this order include EIN2 and EIN3 named after EIN (ethylene insensitive) mutants which are also blocked in their ethylene responses. The EIN2 gene encodes a protein, which may act as a ion channel. The EIN3 gene encodes a protein, which possibly acts as a transcription factor favouring gene expression.
(ii) Ethylene and Regulation of Gene Expression:
It has been observed that the expression of various target genes is altered by ethylene. Ethylene has been shown to increase the levels of mRNA transcripts of several genes corresponding to the enzyme proteins like cellulase, chitinase, β-1, 3-glucanase, peroxidase and chalcone synthase.
Ethylene also increases the expression of genes that encode a pathogenesis-related (PR) protein, genes related with ripening process as well as genes for ethylene biosynthesis.
From the ethylene- regulated PR genes, regulatory sequences called ethylene response elements or EREs that confer ethylene sensitivity to a promoter have been identified. In tobacco, four proteins were identified which are called ERE-binding proteins (EREBPs) that bind to ERE sequences of DNA.
The genes that encode these proteins, viz., EREBPs, may be regarded as ethylene primary response genes. The products of such primary response genes are thought to regulate the expression of secondary response genes, such as the PR genes. Ethylene treatment causes a substantial increase of these EREBPs.
In developing tomatoes, the gene expression related to ripening has been studied extensively. Several genes involved in ripening phenomena are regulated by ethylene. During tomato fruit ripening, the fruits soften by cell-wall hydrolysis by the action of polygalacturonase, green colour changes to red colour caused by chlorophyll loss by chlorophylls together with the synthesis of the carotenoid pigment lycopene.
Apart from this, genes involved in aroma and flavour are also likely to be regulated by ethylene.
The term pathogenesis-related (PR) protein is now used to include all antifungal and antimicrobial plant proteins that are induced following pathogen attack or other stress exposure. PR proteins can also be induced by ethylene and salicylic acid and by a few other growth regulators. In general, PR proteins are resistance factors.
It follows, therefore, that the development of disease-resistant crops seems possible by manipulation of the genes encoding these proteins. Examples of classic tobacco PR proteins are β-1,3-glucanase and chitinase.
Since β-1,3-glucan and chitin, the substrates for these enzymes, are the major components of the cell walls of higher fungi, one can expect that the development of these enzymes in transgenic higher plants serves a defensive function against fungal attack.
(iii) Biochemical Action:
It is well known that applied ethylene induces its own synthesis which is termed as ‘autocatalytic’ ethylene synthesis. In this model, the mode of action of ethylene at the molecular level involves the regulation of ACC synthase. The key enzyme in the biosynthetic pathway, ACC synthase may either be activated or de novo synthesis of this enzyme may be induced by ethylene.
(iv) Ethylene Analogues and Ethylene Antagonists:
Only unsaturated hydrocarbons (olefins) exert ethylene-like biological activity. A compound with a double bond like ethylene is much more active than acetylene having a triple bond. The activity is related to the molecular size, for example, propylene is less active than ethylene. Substituents like halogens lower the activity as in vinyl fluoride and vinyl chloride.
There are two known antagonists that may be applied exogenously to inhibit ethylene action. CO2 prevents or delays many ethylene responses when ethylene concentration is low.
Here CO2 possibly acts as a competitive inhibitor of ethylene action by competing with ethylene for the binding site. High concentration of CO2 delays the ripening action of ethylene and this property is used commercially for the storage of fruits in CO2 atmosphere.
A more potent inhibitor than CO2 is the Ag2 + ion which is thought to block ethylene action by interfering with ethylene binding. Silver salts applied as silver nitrate (AgNO3) or as silver thiosulphate [Ag(S2O3)2] can be used commercially to prolong the vase-life of cut flowers.