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Wednesday, April 23, 2014

WSU-TFREC/Postharvest Information Network/Ethylene in Fruit Physiology



Ethylene in Fruit Physiology


Introduction

The apple is a remarkable fruit. When harvested at optimum storage potential, it may store for as long as 12 months under proper temperature and atmospheric conditions. Correct harvest timing is based on defining the stage of maturity of the apple, which may be best determined by measuring fruit ethylene biosynthesis. Ethylene was discovered in the late 1800's. Like many other discoveries, it was somewhat accidental. Someone had observed several trees along a tree-lined boulevard had lost their leaves. It was soon discovered that there was a leak in one of the underground pipes carrying natural gas. It didn't take long to learn ethylene was the causative agent. Since then, hundreds of papers have been written on ethylene action, biosynthesis, promotion, inhibition, and molecular genetics.

Ethylene is found in most living tissues. In most terrestrial mammals, small amounts of ethylene are expressed with every exhaled breath, but in animals, ethylene is not considered a hormone as it is in plants. There are seven major effects of ethylene in plants which are: promoting ripening, inducing fruit abscission, inducing flowering, promoting seed germination, breaking dormancy, promoting root initiation and inducing vegetative dwarfing. In the science of fruit growing, most of these effects are put to use for the grower's benefit. This paper gives an overview of ethylene's involvement in the process of fruit ripening, how it is measured, and what the measurements mean.


Fruit Development

Ethylene production is largely pre-determined in both time and amount by the genetics of the fruit and, depending on when the fruit blossoms and the climate, will determine to a large degree the development of the respiratory climacteric. During fruit development, respiration (i.e., the generation of carbon dioxide), which is a measure of metabolic activity, declines gradually throughout the season until several weeks before it ripens where it reaches what is known as a preclimacteric minimum. At this point, the metabolic functions of the fruit are in a near resting stage in preparation for a burst of metabolic activity signifying ripening. During ripening both carbon dioxide and ethylene increase significantly. The main developmental stage of the fruit is referred to as maturation, in which photosynthate is converted to starch. The ripening phase is when the starch is converted to sugar. Senescence is that stage in which the membrane functions break down due to degradation of lipid bilayers leading to cell damage and necrosis.

Optimum harvest is a subjective measurement defined as a fruit with good keeping quality and good eating quality. If picked too early during the maturation stage, insufficient starch will be converted to sugar and the fruit will keep well enough, but the eating quality will be poor. On the other hand, if the fruit is picked too late, there will be insufficient starch and acid reserves for metabolic maintenance in storage, but the eating quality will be good. Therefore, the optimum timing is critical to the proper storage (and marketing) of apples. Researchers throughout the world have spent decades defining the optimum time of harvest for long-term CA storage and yet, as new varieties enter the marketplace, optimums must be redefined for each individual cultivar.


Ethylene Production

After the respiratory preclimacteric minimum comes the respiratory climacteric. This is the point at which the fruit will generate a high, sustained respiration and ethylene biosynthesis. It occurs at different times for different cultivars. For example, a Yellow Transparent apple, which ripens in early July, has a very quick respiratory climacteric and may ripen within a few days. In fact, there are times when I have gone out one day and seen green fruit on the tree and two days later, these fruit are yellow and on the orchard floor. The respiratory climacteric of this apple is quick and peak is high. On the other hand, a Delicious apple, which ripens sometime in mid-September, has a somewhat longer climacteric period. This fruit may develop a respiratory climacteric over a period of 10 days, for example. This is advantageous to a grower because it allows time to harvest all of the fruit without being so concerned that the fruit will ripen early and even drop from the tree. Further, there are cultivars that ripen later, such as Fuji, Braeburn, or Granny Smith. These cultivars in turn have an even broader climacteric and the peak of respiration (when similarly measured) is lower than in the previous scenarios.

Consider, for example, ethylene from the cultivar Braeburn. In one of our trials, fruit was picked over a period of 10 weeks beginning around August 15. If we measure the ethylene from the core by sampling with a syringe 24 hours after the fruit has been harvested, we see a very low ethylene production within the fruit tissue--generally less than 2 ppm. On the other hand, if we wait for four days before sampling the core gases of the fruit, we see significantly higher levels of ethylene reaching about 10 ppm at proper harvest. Waiting for seven days after the fruit is harvested before sampling, results in ethylene values in the 60-70 ppm range at harvest time. This suggests that delaying cooling (storage) of the fruit after it has been picked hastens the ripening process and, therefore, is deleterious to the condition of the fruit out of storage. This is important because often times a grower will harvest his fruit in bins and it may stay in his orchard or on the warehouse receiving dock before it is properly cooled. The longer the delay between harvest and storage the shorter will be the storage life and the poorer the condition of the fruit after it is removed from storage.


Sampling Fruit Ethylene

Clearly, the accurate measurement of ethylene in apples is vital to their proper storage and therefore their condition out of storage. But with so many fruit (100 million boxes of roughly 100 apples each in Washington State alone, and at least 4 times that throughout the world) the problem has become not the accuracy of sampling, but the unfathomable number of samples to assess.

Early efforts to analyze gas samples used a bulk approach. A bushel of apples would be placed in a large glass container and a sample of the headspace gas would be withdrawn and sampled. If each apple were very similar, this approach would accurately depict the physiological state of the individual fruit and, therefore, the proper regime for optimum storage. This is hardly the case. Data obtained from this method was qualitative, however, because there always exists the possibility that one apple could be riper than the others, thereby generating most all the ethylene. Thus, although all of the other fruit could have extremely low ethylene levels, the single mature apple would indicate the "average" ethylene was quite high. Because there are differences due to size, color, tree position, stress, variety, rootstock, soil, and a host of other factors, the best that can be said of this method is that it would always overestimate the average ethylene production.

Individual apples were then evaluated for ethylene production. The most common method was to insert an 18-gauge needle into the apple through the calyx end extending through to the core. A 0.5 to 1.0 mL sample of gas could be withdrawn and subsequently analyzed by gas chromatography. (Routine analysis of ethylene by megabore gas chromatography is sensitive to the ten parts per billion range.) By increasing the number of samples and examining the variability among samples, one could obtain a more accurate picture of the physiological state of the part of the orchard sampled. Harvest decisions could then be based on both the amount of ethylene being produced as well as on the degree of variation of samples. High ethylene blocks (experimental sites within and orchard) with high variability might well indicate that very few fruit are producing significant amounts, whereas, high ethylene blocks with little variability might indicate the entire block is ripening more evenly. The concern with this method lies in not knowing the physiological meaning of a gas sample taken from the fruit center. Again, there are differences due, for example, to size of seed cavity, amount of water in the fruit, amount of interstitial space, cell density and the amount of wounding from the needle insertion. Generally, any damage to the fruit tissues induces ethylene biosynthesis.

The method presently under design of assessing individual fruit ethylene is to place each apple in a small, 3-liter Plexiglas chamber with a low flow of scrubbed air. Micro-processors may sample the effluent gas as often as required 24 hours a day. This takes a single person about 1 hour to set up the system capable of analyzing gas composition of hundreds of apples--a process that manually takes about 2 to 3 minutes per sample. The greater number of samples increases the likelihood that apples will reach the consumer with optimum quality.

What is needed is a method to assess quickly and accurately the ethylene generated by the whole apple, or one that uses part of the fruit that is most directly correlated with the physiological stage of fruit maturity. As for sampling a portion of tissue, it almost seems as though Heisenberg's principal is at work here in that any invasive action to the fruit will change its physiological stage and response.

Perhaps the closest thing the apple industry has to assessing individual fruit maturity is in the fruit sorting process. In modern fruit packing warehouses individual fruit are mechanically sorted into weight categories and optically scanned for color sorting. Not far away are sensors for inline determination of firmness, sugar content and internal disorders. Practically everything can be determined about an individual fruit without damaging it. So far, however, only ethylene gives a meaningful picture of the physiological stage of ripeness. Thus, the problem remains; how does the apple industry nondestructively assess ethylene of 10 billion apples.


Inhibiting Ethylene Biosynthesis

Abbott Laboratories has recently developed a new chemical tool for use in apple production. AVG (aminoethoxyvinylglycine) was discovered in 1976 by researchers at Hoffmann-LaRoche, but it wasn't until the last few years that it was made commercially available. This compound is a substituted amino acid produced by a streptococcal mold and is specifically targeted to inhibit the enzymatic production of ACC, which is the precursor of ethylene. In 1980, Williams in Wenatchee showed that AVG could be used to inhibit internal ethylene production thereby reducing fruit abscission, but could not be used to compensate for external ethylene, no matter the source. In 1981, we did some experiments on Golden Delicious apples, in which we took fruit that was harvested at optimum harvest time and dipped them in solutions containing 0, 100, 200, or 400 ppm AVG (Curry, et al.). These fruit were stored in Plexiglas chambers and the ethylene and carbon dioxide were measured daily for a period of seven weeks. Ater about 10 days, the untreated fruit showed an increased rate of respiration that coincided with the climacteric rise. For this treatment, ethylene exceeded 50 ppm for the remainder of the study. Fruit dipped in 100 ppm AVG had a respiratory climacteric delayed about six weeks. At seven weeks, ethylene levels were about 20 ppm and by the ninth week, ethylene had not exceeded 30 ppm. At 200 ppm, it took approximately nine weeks to see a slight increase in carbon dioxide and at that time, ethylene was approximately 4 ppm. Lastly, at 400 ppm, Golden Delicious apples dipped after harvest never showed an increase in respiration and the ethylene levels were near zero, even at nine weeks after harvest. At the end of the nine weeks, the control apples were yellow and shriveled whereas apples dipped in AVG were still green and noticeably firmer.

In another trial, Williams (1980) treated Delicious apples with pre-harvest applications of AVG approximately three days before harvest. These fruit were stored in regular cold storage and after three weeks, showed no difference from those that were not treated with AVG. By 16 weeks, however, differences began to occur. Firmness of untreated apples was 13.9 pounds, whereas those treated with 450 ppm AVG three days before harvest showed an average firmness of 15.2. Thirty weeks after harvest control fruit had a firmness of 12.8 pounds, whereas those treated with AVG were a full pound firmer.


Low Ethylene in Controlled Atmosphere

Controlled atmosphere was developed to provide for the storage of apples as production increased throughout the United States and the world. Controlled atmosphere that pertains to fruit storage is defined simply as the regulation and control of major gases, such as oxygen and CO2, and minor gases, such as ethylene or other volatiles. This is in contrast to an uncontrolled atmosphere or regular storage where none of the gases are regulated, but may be monitored. This atmosphere also changes composition as the apples continue to respire, but it depends entirely upon the venting of the room. Before controlled atmosphere storage, apple storage was limited to the time that the apple would remain marketable (firm) in regular cold storage. This might be either in refrigerated units or natural caves in countries where energy is limited and therefore expensive. With new technology, however, apples may be stored for as long as 12 months. This complicates a grower's life in that in the 12th month of storage, an apple producer has to be considering three apple crops. The first would be the crop still in storage after 12 months. The second would be the current crop that is now on the tree waiting to be harvested. The third would be that which is on the tree in the form of a bud, which has a primordial flower and which is capable of sustaining winter injury. That's a lot of apples to consider at any one time.


Conclusion

In this brief paper, I have tried to indicate why ethylene is one of the main physiological parameter of apple development that must be considered for optimum fruit storage. The implications of ethylene measurement and regulation for fruit and storage are now being realized. Researchers with ARS have genetically engineered a tomato called Endless Summer, in which tissues have a reduced capability of ethylene synthesis and therefore ripen more slowly. More recently, another group of ARS scientists is advancing the pursuit of fruit that has selective tissue sensitivity to ethylene. This could result, for example, in the development of fruit in which only the hypodermis or epidermis would ripen producing enhanced color and flavor or aroma volatiles, but whose cortex would be much less sensitive to ethylene and therefore ripen much less slowly. This might lead to an apple which would ripen on the exterior, and be firm on the interior. Or perhaps we could turn the signal off completely and ripen by external ethylene only. Indeed, if it can be conceived, it can be accomplished. As our technology improves and our understanding of the ripening process increases, we may be able to develop fruit that will ripen exactly how and when we want them to.


References

Williams, M.W. 1980. Retention of fruit flesh firmness and increase in vegetative growth and fruit set of apples with aminoethoxyvinylglycine. HortScience 15(1): 76-77.

Curry, E.A. and M.E. Patterson. 1993. Controlling ethylene biosynthesis with natural compounds. Proc. Wash. State Hort. Soc.: 312-313.

Dr. Eric A. Curry, Plant Physiologist

USDA, ARS Tree Fruit Research Laboratory
1104 N. Western Ave., Wenatchee, WA 98801
CURRY@tfrl.ars.gov

14th Annual Postharvest Conference,
Yakima, Washington
March 10-11,  1998

Tree Fruit Research & Extension Center, 1100 N Western Ave, Washington State University, Wenatchee WA 98801, 509-663-8181, Contact Us