The discovery of ethylene

Never underestimate what a student can accomplish. Dimitry Neljubow was a grad student at the Botanical Institute of St. Petersburg, Russia in 1901. He was trying to grow peas in the laboratory and noticed that they did not grow normally. They were very short, the stems were large in diameter, and bent sideways. This was later called the triple-response of peas.

He discovered that what was causing the abnormal morphology was the air in the laboratory. It turns out that the laboratory was using coal gas (aka illuminating gas) for lamp light. The active ingredient in the air that caused this growth response was ethylene, a byproduct of goal gas combustion.

This was deemed to be simply a pharmacological response until Cousins reported ethylene production in oranges in 1910. This would cause bananas to ripen prematurely if oranges and bananas were stored together. Later careful work showed that the oranges probably did not produce the ethylene, but that Penicillium mold on the oranges probably did. By 1934 ethylene was finally classified as a plant hormone. However, many people considered ethylene production a byproduct of excessive auxin content...a sort of side-effect of a real hormone. Of course this was just an auxin-chauvinistic concept.

The structure and metabolism of ethylene

Below is the structure of ethylene, and some derivatives of ethylene, as well as the breakdown (oxidation) pathway for degrading ethylene. Of course, this pathway is abiotic and so not particularly germaine to plant physiology. Ethylene is a gas, so loss to the air accomplishes its disappearance...not metabolic degradation! The diagram below is chemistry rather than biology.

As you can see ethylene is of low molecular weight and size, it is a gas at room temperature. It is flammable and easily oxidized all the way to carbon dioxide when burned in air. Its double bond makes it reactive, and potent oxidizers like potassium permanganate (KMnO4) can eliminate it from the gas in a room. Activated charcoal can adsorb it too. So for storing fruit, these are some important room-air considerations needed to keep fruit from ripening prematurely.

The biosynthesis pathway discovered by Yang in the 1980s

Work started by Shang Fa Yang in the late 1970s bore fruit in the 1980s as the pathway for biosynthesis of ethylene gas became known. This pathway is partially illustrated below.

The first portion above, has been called the Yang cycle in honor of Shang Fa Yang who died in 2007. He was a very humble and kind man but his contributions to the study of ethylene biosynthesis were amazing. You will notice that the amino acid methionine figures prominently in this pathway. It is perhaps the "entry" point for this pathway. When methionine is conjugated to adenosine, the product S-adenosyl methionine (SAM) is the "exit" of this cycle. What is leaving the cycle is the amino-butyrate portion. You can see it making its exit to the right. The rest of the Yang cycle is the regeneration phase... reworking the methylthioadenosine remainder of SAM back into methionine. The Yang cycle, however is NOT the end of the Yang discoveries...and you might guess from the fact that there is no ethylene shown above!

Ethylene Biosynthesis: the rest of the story

Yang and his students also discovered what happens to the amino-butyrate portion of SAM. The key enzyme here is ACC synthase, which cleaves the amino butyrate from SAM, releasing 1-aminocyclopropane-1-carboxylic acid (ACC). Prior to its discovery by Yang, this molecule was unknown to biologists.

The ACC is then converted to ethylene by ACC oxidase (formerly known as the ethylene forming enzyme). This cleaves the carboxylic acid off as carbon dioxide and its neighboring carbon with the amino group as cyanide gas. That leaves just the two carbons attached to each other with the double bond in the form of ethylene gas. As I am sure you can recognize, then, Yang has elucidated the complete synthesis pathway of ethylene from the amino acid methionine. This is a remarkable accomplishment for one person and his colleagues.

Moreover, you can see that while the plant cell loses its grip on the chemistry as ethylene gas is transported in the atmosphere, Yang realized that the hormone biology homeostasis (transport, conjugation, compartmentation, etc.) considerations must now focus more upon ACC than upon ethylene. This led Yang to elucidate how ACC can be form malonyl-ACC. Whether or not ACC can be retrieved from malonyl-ACC remains to be clearly demonstrated. It does seem that malonyl-ACC simply accumulates in tissues.

Yang's work also made the rest of the plant physiology community realize that the pivotal factor in ethylene production is the amount of ACC in a tissue of interest. The relationship between ACC synthesis and ethylene synthesis has been shown in many examples since Yang's elucidation of the pathway. Below is an example for apple fruit ripening.

As you can easily see, the ethylene synthesis precursor and enzyme are correlated with ethylene production.

Once the gene for ACC synthase was isolated, it was then possible to insert that gene backwards (in the antisense orientation) behind a consitutive (always-on) promoter using Agrobacterium as the intermediary vector. This was done in tomato. The antisense gene produced vast amounts of antisense mRNA; this hybridizes with the sense mRNA coming from the normal gene expression of tomato genes. The double-stranded mRNA is then depolymerized by nucleases into nucleotides. Thus the ethylene signals normally generated by the tomato plant are effectively silenced. Now the big green tomatoes that develop on the plant are not able to produce ethylene and therefore cannot ripen. The green tomatoes are easily stored for a long time, and shipped without damage to stores. However, after these green tomatoes are gassed with ethylene, they ripen just as normal. So this permits tomatoes to be sold in near-perfect condition at just about any time of the year.

Ethylene and Fruit Ripening

In addition to its role in "slow-ripening" tomatoes, ethylene is widely known to be involved with initiating a ripening process in a range of fruits, especially those which show a rapid rise in respiration rate just before ripening. This rapid increase is called the climacteric. Examples of fruits which show this include:

Climacteric Not Climacteric
Bell pepper
Snap Bean

The relationship between ethylene production and this climacteric rise in respiration is shown below.

Obviously the ethylene signal appears to stimulate the respiration rise...this is the case in banana. The bananas you get from the store have usually been gassed with ethylene already, so their climacteric is already underway and they will ripen quickly after you get them out of the store. Putting them in the refrigerator helps slow this process, but it also turns the skin a dark brown. The fruit tissue inside, however, will remain firm for much longer in the refrigerator than it does out at room temperature.

After this climacteric, a range of senescence events follow. Enzymes are produced to change the qualities of the fruit. A green (unripe) fruit has a suite of characteristics depending on the species. The skin is green, the fruit is hard, it is loaded with starch, it is acidic, it is odorless. The climacteric respiration increase signalled by ethylene, supports the transcription and translation of genes for hydrolytic enzymes. Some break down chlorophyll, some synthesize anthocyanins, some digest starch into sugar (amylase), some digest pectins (pectinase) that glue the cells together, softening the fruit. Other enzymes convert organic acids into neutral pH compounds (the tartness goes away). The sugar from starch breakdown becomes a solute and water enters the fruit from the xylem. Other enzymes degrade odorless large organic molecules into small aromatic ones. The green, hard, mealy, odorless fruit changes to a bright color, sweet, juicy, soft fruit with a species specific odor.

My favorite example for fruit ripening, is pear. These fruits are sold rock-hard in the store and are often served in college cafeterias rock-hard. Of course it is a matter of taste whether you would want to eat the pear in that state...certainly not my choice. People who put their pears in the refrigerator when they get home from the grocery will keep the pears in the hard-state for long periods of time. Rather than improving, the pears just take on "refrigerator odors," degrading their quality. What should be done with pears and is seldom done in homes and restaurants and cafeterias is to leave them out at room temperature! Leaving them in a paper bag to allow gas exchange while maintaining an atmosphere inside the paper bag of higher ethylene accumulation will allow the pears to ripen. Another good approach is to put the pears in a fruit bowl on the dining table for several days. The warm temperature and access to oxygen allows the pears to go through their climacteric, to let the ethylene stimulate the transcription and translation of enzymes of ripening described above. That tasteless, rock-hard pear turns into a luscious, sweet, butter-soft, juicy fruit with an incredible aroma and flavor that only those who know the secret to ripening pears have savored.

Fruit ripening is a case of general senescence

The same sequence of events involved in fruit ripening, take place in leaf senescence too. The same ethylene signal, respiration rise, and degradation of complex organics into simpler ones are shared processes. When we consider that a fruit is a carpel, a carpel is a modified leaf, then it may come as no surprise that the leaf senescence process is the same process used for fruit ripening.

The hypothesis you tested in lab was that auxin produced at low concentration by a blade and/or cytokinins coming up from the roots maintain the integrity (similar to fruit hardness) of the leaf. Its color is green and the abscission zone (where the petiole attaches to the stem) is composed of cells glued firmly together with pectins. But when the days get short (nights get long!) and the nights are much colder than the days, the plant initiates senescence. Ethylene production stimulates respiration and the gene expression for enzymes. These enzymes degrade chlorophyll and the Magnesium and Nitrogen and Phosphorus are loaded into the phloem and put into the trunk of the trees for winter. Cheap pigments (hydrocarbons mostly) such as anthocyanin and carotenoids are left in the leaf. So people flock to New England to view the beautiful change of color in our trees in mid-October.

It is also true that leaf senescence in the autumn is repeated in the leaf petiole to lead to leaf abscission. This too is just another example of the senescence protocol. However, it is magnified in a layer of cells at the base of the petiole. These cells are signalled by ethylene, their respiration rises, they produce enzymes including pectinase, the pectinase unglues the cells in the abscission zone, and the leaf falls from the tree. This zone of specially-responding cells is called the abscission zone.

Again, if a fruit is a carpel and a carpel is just a modified leaf, then you would be right to expect that fruit abscission works just like leaf abscission, only it has more weight to pull on the abscission zone. And perhaps Newton discovers gravity?!

The understanding of how flowers work becomes apparent. This was the thesis of my PhD disseration back in 1981. Each petal and stamen is also a modified leaf. Right now the apple and pear trees are in bloom, but these flower parts lose their ability to maintain IAA production, the parts are not maintained, and ethylene signals senescence and abscission. The petals and stamens shower around us. The carpel, however, contains seeds which produce hormones necessary to maintain the carpel on the plant. These overcome any senescence signals sent out by stamens, petals, and sepals nearby; just as some of your treatments of wheat leaves in lab inhibited senescence. In another project we debladed some primary leaves in kidney bean (Phaseolus vulgaris) in lab and found that the loss of IAA signal causes the petioles abscise rapidly thereafter. Adding a dollop of lanolin with IBA in it to the petiole stump should have caused the petiole to remain awhile longer (if we got the IBA concentration right...right?!). What was our hypothesis with cytokinin application?

Ethylene has a range of roles

In addition to the triple response (epinasty, lateral expansion, dwarfing), fruit ripening, fruit abscission, leaf senescence and leaf abscission, ethylene also simulates seed germination in some species, maintains hypocotyl hooks in seedlings, stimulates stem growth in underwater species, stimulates root hair growth and induces root formation, and promotes flowering in pineapples! As is the case for many of the plant hormones, ethylene has pleiotropic effects.

A mechanism for ethylene responses

An idea of how ethylene may regulate the production of respiration enyzmes, fruit ripening and abscission enzymes, growth effects and so on, is being elucidated at the level of the central dogma. The common thread in the findings to date are membrane receptor proteins interacting with intracelular secondary messengers, influencing transcription factors, turning "on" certain genes in the genome, and eliciting particular reactions in response. There is one interesting twist in the case of the ethylene signalling pathway.

The ethylene cell-membrane receptor protein appears to include a copper cofactor and can also be eliminated when the receptor binds to silver (Ag+) ions usually supplied as silver thiosulfate. The silver thiosulfate can thus be added to a vase to increase the vase-life of a bouquet of flowers.

Also, one could imagine that a defective ethylene receptor from a mutant ETR1 gene could produce "never-ripen" tomatoes...even ethylene gas would not permit them to ripen. Yup, we have made such mutant tomatoes...the better to add to your BIG MAC...yuck! While we might not appreciate those genes in our sandwich, we might like those mutant ETR1 genes in our roses. I imagine those are coming but I haven't seen them yet.