Ethylene (C2H4) is a gaseous hormone produced from bacteria, fungi and all parts of higher plants such as shoots, flowers, seeds, leaves, roots, and fruits (Pech et al., 2003, p. 247). Ethylene is a two carbon symmetrical compound with one double bond (CH2=CH2). Its molecular weight is 28.05, with freezing, melting, and boiling temperatures of -180°C, -169.5°C and -103.7°C respectively. It is a flammable and colourless gaseous compound (Arshad & Frankenberger, 2002, pp. 1-9).
The recognition of ethylene as a plant hormone originated from the historical observatory facts of premature shedding of street trees, geotropism of etiolated pea seedlings when exposed to an illuminating gas, early flowering of pineapples treated with smoke, and ripening of oranges when exposed to gas from kerosene combustion (Arshad & Frankenberger, 2002, pp. 1-9). Neljubow (a Russian graduate) in 1901, demonstrated first time that ethylene is involved in the above mentioned responses of plant (Reid, 2002, p. 149). Later, in late 1950 the invention of extremely sensitive technique involving gas chromatography has established a conclusive concept that ethylene is a naturally occurring endogenous plant hormone. It has numerous but complex interactions with other plant hormones, particularly with auxins (Hyodo et al., 1984; Arshad & Frankenberger, 2002, pp. 1-9).
Ethylene affect various aspects of the growth and development of plants (Pech et al., 2003, p. 247, ). Initially it was only regarded as a 'ripening hormone' but later investigations have proved its importance as a plant hormone, playing diverse and effective role in many plant physiological developmental processes. The major areas of plant physiology in which ethylene have been known to influence plant growth and development, includes release of dormancy, shoot-root growth & differentiation, adventitious root formation, flower induction in some plants, induction of femaleness in dioecious flowers, leaf and fruit abscission, flower ripening, flower & leaf senescence and fruit ripening (Arshad & Frankenberger, 2002, pp. 1-9).
Moreover, being a ripening hormone ethylene play a very important role in the postharvest life of many horticultural products, like increasing senescence speed and reducing shelf life but beneficially it improves the quality of the fruit and vegetables by manipulating uniform ripening process (Reid, 2002, p. 149). Because of the enormous influence of ethylene on the physiological development and postharvest life of fruits and vegetables, its biosynthesis, action, and control have been intensively investigated (Reid, 2002, p. 149; Pech et al., 2003, p. 248).
ETHYLENE PRODUCTION
In fruits and vegetables several metabolic reactions starts after harvesting. In most cases, an increase in biosynthesis of gaseous hormone like ethylene serves as the physiological indication for the ripening process. During ripening process, in some fruits large amount of ethylene is produced which is usually referred as a 'autocatalytic ethylene' production response. However, fruits are divided into two main categories on the basis of ethylene production, i.e. climacteric (those produce large amount of ethylene) and non-climacteric fruits (those produce smaller amount of ethylene). In climacteric fruits like apple, pear, banana, tomato and avocado, ethylene production usually ranges from 30-500 ppm/(kgh) during ripening. While non-climacteric fruits like orange, lemon, strawberry and pineapple, produce 0.1-0.5ppm/(kgh) of ethylene (Paliyath & Murr, 2008a) (Table 1). Therefore application of even a very low concentration of ethylene (0.1-1.0 μL/L) is sufficient enough to accelerate full ripening of climacteric fruits; however, the magnitude of the climacteric rise is not dependent on the amount of ethylene treatment (Figure 1 & 2). On the contrary, application of ethylene causes a temporary rise in the rate of respiration of non-climacteric fruits, and the magnitude of the increase is dependent on the amount of ethylene (Biale, 1964, as cited in Wills et al., 1998, pp. 106-107).
In addition, the difference in the respiratory patterns of climacteric and non-climacteric is associated with the different behaviour in terms of the production and response to ethylene gas (Burton, 1982). The increase in respiration, as influenced by ethylene application, may happen several times in non-climacteric fruits, but only once in climacteric fruits (Biale, 1964, as cited in Wills et al., 1998, pp. 106-107).
Ethylene is also produced from storage or transport containers. Usually the gases produced from petroleum combustion engines of forklifts or vehicles are also a source of ethylene and cause contamination of the stored products (Wills et al., 1998, pp. 106-107)
Table 1: Classification of fruits and vegetables according to the ethylene production rates at optimum handling temperatures
Relative ethylene production rate (µL/kg/hr)
Fruits
Vegetables
Very low (less than 0.1)
Cherry, Strawberry
Artichoke, Asparagus, Beets, Cabbage, Carrot, Cauliflower, Celery, Cherry, Garlic, Leeks, Lettuce, Onion, Parsley, Parsnip, Peas, Radish, Spinach, Sweet Corn, & Turnip
Low (0.1 to 1.0)
Blackberry, Blueberry, Kiwifruit (unripe), Persimmon, Raspberry
Broccoli, Brussels Sprouts, Endive, Escarole, Green Onions, Mushroom
Moderate (1.0 to 10)
Fig, Banana, Lychee, Mango, & Plantain
Melons, tomato
High (10 to 100)
Apples, Apricot, Kiwifruit (ripe), Nectarines, Peach, Pear, Plum, Avocado, Feijoa, & Papaya
-------
Very High (above 100)
Cherimoya, Passion fruit, & Sapote
-------
Source: Kader & Kasmire, 1984
Figure 1: Effect of ethylene on Banana ripening (Source: Morrelli & Kader, 2006)
Figure 2: Effect of ethylene different concentrations on Persimmon fruit (Source: Crisosto et al., 2006)
ETHYLENE BIOSYNTHESIS
Indeed, ethylene is produced by all parts of the plant but the magnitude of ethylene production varies from organ to organ and also depends on the stage and type of growth and developmental process. In fact, recent ethylene based research findings have increased the understanding of biosynthetic pathways and enzymes involved in ethylene production, as well as the development of several ways to manipulate ethylene production e.g. by genetic alteration of plants (Arshad & Frankenberger, 2002, p. 11). Ethylene is produced by various plant parts growing under normal conditions however, any kind of biological, chemical or physical stress (e.g. wounding) strongly promotes endogenous ethylene synthesis by plants. Among stress induced ethylene production, pre-harvest deficit irrigation is one of the most important factor causing higher ethylene production rates in fruits like avocado (Adato & Gazit, 1974) and tomato (Pulupol et al., 1996).
In fact, the credit of discovering the ethylene biosynthetic pathway goes to Shang Fa Yang and his co-workers (Arshad & Frankenberger, 2002, p. 11). The biosynthetic process of ethylene is usually completed in three major steps. Along with a schematic representation of the ethylene biosynthetic pathway is given in the figure 3.
Step I:
The biosynthesis of ethylene hormone is started by the conversion of Methionine (MET) to S-adenosyl-L-methionine (SAM) by the enzyme methionine adenosyltransferase (Pech et al., 2003). However, methionine adenosyltransferase is thought to consider as a rate limiting enzyme in ethylene biosynthesis because formation of SAM depends on the activity of this enzyme and SAM levels may indeed regulate ethylene production. Therefore, the sensitivity or importance of methionine adenosyltransferase to SAM implies that this enzyme may play a regulatory role in ethylene biosynthesis (Arshad & Frankenberger, 2002, p. 13).
Stage II:
SAM is consequently converted to 1-aminocyclopropane-1-carboxylic-acid (ACC) by a pyridoxal enzyme ACC synthase (ACS) (Figure 3). Actually, before the discovery of ACC, as intermediate, immediate precursor in MET dependent ethylene production process, the ethylene biosynthetic pathway was intangible (Arshad & Frankenberger, 2002, pp. 11-50). The conversion of SAM to ACC by ACS is another rate-limiting step in the biosynthetic pathway of ethylene. ACS is a cytosolic enzyme (found in the cytoplasm of plants) (Paliyath & Murr, 2008b) and its activity is strongly inhibited by aminoethoxyvinylglycine (AVG) (a competitive inhibitor) and aminoisobutyric acid (AIB) (an inhibitor of pyridoxal phosphate-mediated enzyme reactions) (Arshad & Frankenberger, 2002, pp. 11-50). Moreover, the activity of ACC synthase is also influenced by factors such as fruit ripening, senescence, auxin levels, physical stresses, and chilling injury. The synthesis of this enzyme increases with an increase in the level of auxins, indole acetic acid (IAA) and cytokinins (Wills et al., 1998, p. 42).
Stage III:
At last the ACC converts into ethylene by the action of ACC oxidase (known as 'ethylene forming enzyme' or EFE) (Arshad & Frankenberger, 2002, pp. 11-50; Pech et al., 2003). However, ACC oxidase is a bi-substrate enzyme as it requires both oxygen and ACC. Moreover, this enzyme also requires Fe2+, ascorbate and CO2 for its activity. Activity of ACC oxidase is inhibited by cobalt ions, and temperatures higher that 35oC (Wills et al., 1998, p. 42). The sub cellular position of ACC oxidase is still a point of controversy because there is a large number of data is available showing that this enzyme is associated with plasma-membrane or with apoplast or tonoplast. The activity of this enzyme (ACC oxidase) has been studied in many horticultural crops like melon, avocado, apple, winter squash, pear and banana. The activity of ACC oxidase is not highly regulated as ACS. It is constituted in most vegetative tissues and it is induced during fruit ripening, wounding, senescence and fungal elicitors (Arshad & Frankenberger, 2002, pp. 11-50).
Figure 3. Ethylene biosynthesis in plants. (Source: Wang et al., 2002)
However, it is clear from the ethylene biosynthetic pathway that those biochemical steps involving ACC synthase and ACC oxidase are the key regulatory points in the biosynthesis of ethylene (Paliyath & Murr, 2008b). Furthermore, Koning (1994) has also explained the correlation among ethylene biosynthesis enzymes and ethylene released in figure 4.
Figure 4: Correlation ethylene biosynthesis and enzymes involved (Source: Koning, 1994)
REGULATION OF ETHYLENE BIOSYNTHESIS
Furthermore, in plants there is also a concept that multiple gene are thought to be responsible for the activity of certain enzymes (e.g. ACC synthase and ACC oxidase) involved in the regulatory process of ethylene biosynthesis. Moreover, in plants ethylene itself stimulates the ability of the tissue to convert ACC into ethylene, which is also regarded as phenomenon of 'auto-regulation'. In ripening fruits, regulation of ethylene biosynthesis is a characteristic feature and is triggered by the exposure to exogenous ethylene by the activation of ACC synthase and/or ACC oxidase (Arshad & Frankenberger, 2002, pp. 25-27).
On other hand, sometimes ethylene inhibit its own synthesis as negative feedback has already been recognised in a number of fruits and vegetable tissues. In such cases, exogenous ethylene significantly inhibits the production of endogenous ethylene, induced by ripening, wounding and/or treatment with auxins. Moreover, this auto inhibitory effect seems more directed towards limited availability of ACC in the presence of AVG, an inhibitor of ACC synthase (Arshad & Frankenberger, 2002, pp. 25-27). Scientists have also revealed that the inhibition or negative regulation of ethylene synthesis is the result of activity of a gene, E8 whose expression leads to the inhibition of ethylene production in tomatoes (Arshad & Frankenberger, 2002, pp. 25-27).
MECHANISM OF ACTION
In literature, number of hypothesis are available, explaining the mechanism of action of ethylene. For example,, the concept of ethylene activity either as a cofactor in some reactions, or by being oxidised to some essential components and being incorporated into tissue, or by binding to a receptor and then either by diffusing away or being destroyed. Furthermore, the response of ethylene action can be classified into two categories namely concentration response and sensitivity response. The concentration response involves the changes in concentration of cellular ethylene while the sensitive response involves the increase in tissue sensitivity to ethylene. Moreover, both of these responses involves the binding of ethylene to come components of the cell to mediate the physiological effects (Arshad & Frankenberger, 2002, pp. 28-36).
Wills et al. (1998, pp. 42-45) likewise explained that plant hormones control the physiological processes by binding to specific plant or fruit receptor sites, which trigger the succession of events leading to visible responses. In the absence of ethylene, these receptor sites are active, allowing the growth of plant and fruit to proceed. During fruit ripening, ethylene is produced naturally or, if it is artificially introduced in a ripening room, it binds with the receptor and inactivates it, resulting in a series of events like ripening or healing of injuries in plant organs. Ethylene action can be controlled through modification of the amount of receptors or through disruption of the binding of ethylene to its receptors. Binding of ethylene is believed to be reversible at a site which contains metal like copper, zinc, or iron (Burg & Burg, 1965, as cited in Burton, 1982). The affinity of receptor for ethylene is high in the presence of oxygen and decreases with carbon dioxide.
Changes in the pattern of ethylene production rates and the internal concentrations of ethylene associated with the onset of ripening have been studied in various climacteric fruits. For instance, tomato and honeydew melon exhibited a rise in ethylene concentration prior to the onset of ripening, determined as the initial increase in respiration rate. On the other hand, apple and mango did not show any increase in ethylene concentration before the increase in respiration (Wills et al., 1998, pp. 42-45).
Ripening has been associated with senescence as it leads to the breakdown of the cellular integrity of the tissue. It is part of the "genetically programmed phase in the development of plant tissue with altered nucleic acid and protein synthesis occurring during the onset of the respiratory climacteric resulting in new or enhanced biochemical reactions operating in a coordinated manner" (Wills et al., 2007, p. 40). These concepts confirm the known degradative and synthetic capacities of fruit during the ripening process. The ability of ethylene hormone to initiate biochemical and physiological events leads to the theory that ethylene action is regulated at the level of gene expression (Pech et al., 2003; Wills et al., 1998, pp. 45-46). Furthermore, Alexander & Grierson (2002) have also explained the same concepts about the ethylene action during the tomato fruit ripening (Figure 5).
Ethylene
Ethylene Receptors
Signal Transduction
Altered Gene Expression
FRUIT RIPENING
Autocatalytic Ethylene production
Changes in cell wall metabolism
Carotenoid accumulation
Chlorophyll degradation
Synthesis of volatiles
Increase in sugars & acids
Senescence
Defence Signalling
Figure 5 : Schematic representation of the role of ethylene during fruit ripening.
Source: Alexander & Grierson, 2002
ETHYLENE-RESPONSIVE GENES
Ethylene is involved in the transcription of a number of specific genes during different stages of the growth and development of a plant or under the action of different stimuli. By using different processes, role of ethylene in the regulation of gene expression has been explained. These processes includes, treatment of tissues with exogenous ethylene; inhibition of ethylene action using the hormone antagonists; analysis of mutants impaired in ethylene perception; and characterisation of transgenic plants with low ethylene production (Pech et al., 2003). With these methods, various types of genes have been found to be ethylene-regulated are give as;
Ripening or senescence-related genes: E4, E8, E17, J49, proteinase inhibitor; cellulose; and polygalacturonase.
Pathogenesis-related genes: chitinase, β-1,3-glucanase, hydroxyproline-rich glycoproteins
Wound-induced genes: phenylalanine ammonia lyase, 4-coumarate-CoA ligase, chalcone synthase.
There are a number of mechanisms by which ethylene regulates gene expression (Pech et al., 2003). Early mature-green tomatoes treated with exogenous ethylene show a rapid accumulation of mRNAs related to ethylene-responsive genes. Post-transcriptional processes likewise influence the regulation of gene expression by ethylene. Analysis of ACS-antisense tomato fruit showed that accumulation of polygalacturonase (PG) transcripts is developmentally regulated during the ripening process. PG mRNA, but not PG polypeptide, accumulates in tomato fruit with reduced ethylene production. Furthermore, synthesis of PG proteins takes place when the fruit is treated with ethylene, demonstrating that PG gene expression is regulated by ethylene at the post-transcriptional level (Theologis et al., 1993, as cited in Pech et al., 2003).
Moreover, Davis & Grierson (1989, as cited in Arora, 2008) observed that in tomatoes, the highest level of expression of pTOM genes in fruits was detected at the orange stage when ethylene production was highest. They proved that exogenous application of ethylene results in increased expression of pTOM genes in fruits and leaves, and provided an evidence that gene expression is involved in both fruit ripening and leaf senescence. Furthermore, genes for production of heat shock proteins are also identified during ripening and/or ethylene production (Gray et al., 1992, as cited in Chaves & Mello-Farias, 2006). Some examples of ethylene regulated or enhanced ripening genes are also given in the table 2.
Table 2: Some known examples of ethylene enhanced ripening related genes
Gene
Homology
Function
