Let us make an in-depth study of the ethylene as plant growth hormone. After reading this article you will learn about 1. Discovery of Ethylene 2. Chemical Nature of Ethylene 3. Detection & Estimation of Ethylene 4. Biosynthesis of Ethylene 5. Inhibitors of Ethylene Biosynthesis 6. Antagonists of Ethylene Action 7. Inactivation of Ethylene in Plants 8. Occurrence, Distribution & Transport of Ethylene in Plants and 9. Mechanism of Ethylene Action.
Discovery of Ethylene:
A unique feature about discovery of ethylene as plant growth hormone is that experimenters first knew ethylene as exogenous chemical affecting plant growth and thereafter only gradually and over a period of over half a century, it became evident that it is in-fact a natural plant growth hormone.
The discovery of ethylene as a plant growth regulator may indirectly be traced back to 19th century when street lights were used to be lighted with illuminating coal gas. It had been a common observation at that time that the trees in the vicinity of the street lamps defoliated more extensively than other trees. Even ancient Chinese knew that their harvested fruits would ripen much faster in burning incense. (Ethylene was later identified as important component of coal gas or burning incense which was probably responsible for such effects on plants).
However, the credit for first establishing the fact that ethylene affects plant growth goes to a Russian physiologist Dimitry N. Neljubow who in 1901 identified ethylene in laboratory air from illuminating coal gas which caused typical symptoms in etiolated pea seedlings grown in dark in the lab. viz.,
(i) Inhibition of stem elongation,
(ii) Stimulation of radial swelling of stems and
(iii) Horizontal growth of stems with respect to gravity.
These symptoms were later termed as ‘triple response’ and were not observed in etiolated pea seedlings grown in normal air free from coal gas. ‘The first indication that ethylene might be a natural product of plant tissues came through an annual report by H. H. Cousins in 1910 to Jamaican Agriculture Department wherein he mentioned that “bananas should not be stored with oranges in ships because some emanations from oranges caused bananas to ripen prematurely”.
(However, since oranges produce relatively very little ethylene in comparison to other fruits such as apples, it was probably the fungi such as Penicillium on infected oranges which produced substantial amount of ethylene as emanations). But, it was R .Gane (1934) who clearly established that ethylene is actually a natural product of ripening fruits and is responsible for faster ripening process.
Meanwhile, several other experimenters found evidence of ethylene being produced not only by ripening fruits but also by flowers, seeds, leaves and even roots and having profound regulatory activity in plants. But, their proposals to consider ethylene as natural plant hormone met with strong criticism by other well known physiologists of that time especially Went and Thimann (1937) and were rejected. (The idea was then prevalent that auxin was the main plant hormone and the effects of ethylene on plant growth were considered to be due to auxin and that ethylene played only insignificant and indirect physiological role. The problem was compounded due to lack of suitable techniques for quantification of ethylene in plant tissues at that time).
For further almost two and a half decades, the importance of ethylene as natural plant hormone remained subdued. It was only after the advent of gas chromatography (GC) and its use in ethylene research, that importance of ethylene as important hormonal regulator of physiological processes was realised (Burg and Thimann, 1959, 60). Soon, this was followed by an avalanche of experimental research work on ethylene and finally ethylene emerged as an accepted natural plant growth hormone (Pratt and Goeschal, 1969).
Chemical Nature of Ethylene:
Ethylene (C2H4) with a molecule weight of 28, is a well known and simplest olefmic gas and has the following structural formula,
Ethylene is flammable and highly volatile substance that readily undergoes oxidation to produce ethylene oxide. In many plant tissues, ethylene can be fully oxidised to CO2 through ethylene oxide. It is colourless, lighter than air at room temp, and under physiological conditions and is sparingly soluble in water. Ethylene is readily absorbed by potassium permanganate (KMnO4). The latter is frequently used to remove excess ethylene from the storage chambers.
Detection & Estimation of Ethylene:
Previously, bioassay methods based on etiolated dicot seedlings ‘triple response’ were used to detect and estimate ethylene concentrations. But, these methods have now been replaced with most accurate and sensitive gas chromatographic technique along with the flame ionization detector. This technique is so sensitive and accurate that it is possible to detect as low as 3ppb (3 parts per billion i.e., 3pLL–(3 pico liter per liter); pico = 1012) of ethylene within a few (1-5) minutes only. More recently, laser driven photo-acoustic detector has been in use which can detect as low as 50ppt (50 parts per trillion) i.e., 0.05 pLL– of ethylene.
Biosynthesis of Ethylene:
Ethylene is known to be synthesized in plant tissues from the amino acid methionine. A non-protein amino acid, 1-amino cyclopropane-l-carboxylic acid (ACC) is an important intermediate and also immediate precursor of ethylene biosynthesis. The two carbons of ethylene molecule are derived from carbon no. 3 and 4 of methionine.
Whole process of ethylene biosynthesis (Fig. 17.27) is a three steps pathway and is aerobic:
(i) First Step:
In the first step, an adenosine group (i.e., adenine + ribose) is transferred to methionine by ATP to form S-adenosylmethionine (SAM). This reaction is catalysed by the enzyme SAM-synthetase (methionine adenosyl transferase).
(ii) Second Step:
In the second step, SAM is cleaved to form 1-aminocyclopropane-l- carboxylic acid (ACC) and 5′-methylthioadenosine (MTA) by the enzyme ACC-synthase.
i. Synthesis of ACC is rate limiting step in ethylene biosynthesis in plant tissues.
ii. Exogenously supplied ACC greatly enhances production of ethylene in plant tissues.
(iii) Third Step:
In the third and last step of ethylene biosynthesis, ACC is oxidised by the enzyme ACC-oxidase (previously called ethylene forming enzyme i.e., EFE) to form ethylene. Two molecules, one each of HCN and H2O are eliminated.
i. ACC oxidase activity can be rate limiting step in ethylene biosynthesis in plant tissues which show high rate of ethylene production such as ripening fruit.
ii. The enzyme ACC oxidase requires ferrous iron (Fe2+) and ascorbate as cofactors.
iii. ACC can be conjugated to give N-malonyl ACC (Fig. 17.27) and thus, may play an important role is regulation of ethylene biosynthesis.
There is only limited amount of free methionine (which is a sulphur containing amino acid) in plant tissues. Therefore, to sustain normal rate of ethylene biosynthesis, the sulphur released during ethylene biosynthesis is recycled to methionine again through methionine cycle or Yang cycle (so named after the pioneer worker S.F. Yang on ethylene biosynthesis). The CH3-S group is salvaged and reappear in methionine as a unit. The remaining 4C atoms of methionine are supplied from ribose moiety of ATP which was originally used to form SAM. A transamination reaction provides the amino group (Fig. 17.27).
Factors Stimulating Ethylene Biosynthesis:
Ethylene biosynthesis is known to be stimulated by a number of factors such as IAA, cytokinins, fruit ripening, stress conditions (drought, flooding, chilling, exposure to ozone etc.) and mechanical wounding. In all these cases, ethylene biosynthesis is stimulated by induction of ACC synthase. In climacteric fruits, ethylene itself promotes biosynthesis of ethylene by autocatalysis.
Inhibitors of Ethylene Biosynthesis:
There are two potent inhibitors of ethylene biosynthesis viz., amino ethoxy-vinyl- glycine(AVG) and aminooxyacetic acid (AOA) which block conversion of SAM to ACC (Because AVG and AOA are well known inhibitors of enzyme that requires pyridoxal phosphate as cofactor, the enzyme ACC synthase is a pyridoxal phosphate dependent enzyme.
Cobalt ions (CO2+) are also known to inhibit ethylene biosynthesis by blocking the conversion of ACC to ethylene by ACC oxidase.
Antagonists of Ethylene Action:
A number of inhibitors are known which inhibit ethylene action in plants.
A brief account of these follows:
At high concentration (5 to 10%), CO2 inhibits many effects of ethylene such as induction of fruit ripening. CO2 probably acts as competitive inhibitor of ethylene action. According to Burg (1965), the relative affinity of active site for CO2 and ethylene is 1:100,000.
Because of this effect, CO2 at higher concs. is often used to delay ripening of picked fruits and some vegetables. However, the high conc. of CO2 required for inhibition makes it unlikely that CO2 often acts as antagonist of ethylene action in vivo.
(ii) Silver Ions:
Silver ions (Ag+) in the form of silver-nitrate (AgNO3) and especially as silver this sulphate (Ag (S2O3)3-2 are potent and much more effective inhibitors of ethylene action than CO2. They are known to inhibit triple response of etiolated pea seedlings, enhancing abscission of leaves, flowers and fruits of cotton and induction of senescence in orchid flowers. Silver thiosulphate is much more effective in delaying senescence of cut flowers than silver nitrate.
(iii) Synthetic Volatile Olefin Compounds:
In recent years, many synthetic volatile olefinic compounds have been found to be strong competitive inhibitors of ethylene receptors and thus preventing ethylene action. Some of these are, trans-cyclooctene, 1-methyl cyclopropene (MCP) and 2, 5-norbornadiene.
Inactivation of Ethylene in Plants:
Due to its high diffusivity, excess ethylene can readily be flushed out of plant tissues. Therefore, catabolism of ethylene is not of much significance in plants. Nevertheless, radio isotopic studies done with 14C labelled ethylene have shown CO2, ethylene oxide and ethylene glycol to be the major breakdown products of ethylene in plant tissues. But, these breakdown products are known to play hardly any role in regulating level of this gaseous hormone (ethylene) in plants.
During biosynthesis of ethylene in plant tissues, not all ACC is converted into ethylene and some of it is diverted to form its conjugated form as N-malonyl ACC that accumulates in plant tissues and may play important role in regulating level of ethylene in plant tissues.
Occurrence, Distribution & Transport of Ethylene in Plants:
Ethylene is produced by all groups of plants including bacteria, fungi, some blue-green algae, bryophytes, pteridophytes, gymnosperms and angiosperms. In higher plants, ethylene can easily be synthesized in all plant organs such as roots, stems, leaves, tubers, bulbs, fruits and seeds. But, its production varies depending on the type of tissue and stage of development. It is highest in senescing tissues and ripening fruits.
Within the plant organs, ethylene formation is mainly located in peripheral tissues.
Ethylene is biologically active at very low concentration (<1ppm). According to Kidd & West (1945), the minimum threshold value for many ethylene dependent responses in plants was about 0.1 ppm.
As a by-product of hydrocarbon combustion, ethylene is also a common environmental pollutant that can play havoc with green house cultures and/or laboratory experiments.
Due to its hydrophobic nature, ethylene can easily pass through plasma-membrane into the cell, easily diffuse within the plant or plant part and flushed out of plant tissues through intercellular spaces. Cuticle on plant surfaces acts as resistant barrier, while stomata, lenticels and cut places serve as exit points for the gaseous hormone.
Mechanism of Ethylene Action:
As mentioned earlier, ethylene exhibits wide range of physiological effects in plants. However, earlier steps are assumed to be similar in all cases that include,
(i) Binding of ethylene to a receptor,
(ii) Activation of one or more signal transduction pathways and
(iii) Modulation of gene expression leading to cellular response.
i. In Arabidopsis thaliana and other plants, ethylene responses are negatively regulated by a receptor gene family.
ii. An ethylene binding receptor protein has been identified in A .thaliana, tomato and other plants which is known as ETR1. Four other proteins are also known that serve as ethylene receptors which are called as ETR2, ERS1, ERS2 and EIN4.
iii. Like cytokin receptor, the ethylene receptor (ETR1& others) is also similar to bacterial two component sensor histidine Kinases. It is a dimer of two polypeptides which are held together by disulphide (-S-S-) bonds and is located on ER (endoplasmic reticulum).
iv. Each polypeptide of the dimer receptor protein consists of three domains,
(i) Amino terminal domain which spans the ER membrane at least thrice and contains the ethylene binding site,
(ii) A middle histidine kinase domain and
(iii) A receiver domain towards the carboxyl terminus of the polypeptide (Fig. 17.28).
v. Ethylene binds to trans membrane domain of the receptor through a copper cofactor. The latter is incorporated into the receptor protein by RANI protein.
vi. Binding of ethylene to receptor inactivates a protein kinase such as CTR1 (a member of RAF family of protein kinases) in the cytosol.
vii. Inactivation of CTR1 allows a trans-membrane protein (on ER) called EIN2 to function.
viii. EIN2 now activates a cascade of transcription factors in the nucleus such as EIN3, ERF1 etc.
ix. These transcription factors in turn modulate gene expression and ultimately ethylene response occurs.
(In the absence of ethylene, CTR1 becomes active which inhibits downstream signal transduction components and therefore, ethylene response does not occur).
Various steps of ethylene signalling are shown in Fig. 17.28.