Realising the role of antisense RNA technology in the reduction of ethylene production, a cDNA (pTOM13) encoding ACC oxidase has been isolated from a tomato fruit ripening library. An antisense gene construct from the cDNA strongly inhibited expression of ACC oxidase activity in transgenic tomatoes.
A chimaeric pTOM13 antisense gene, encodes ACC oxidase consisting of 1.3 kilobase (kb) fragment of the cDNA inserted in a reverse orientation between CaM35S promoter and terminator and was transferred to tomato plants using Agrobacterium tumeifaciens as a vector. All transformed plants analysed showed reduced ethylene synthesis.
Interestingly, inhibition of Acc oxidase was accomplished by a single-copy antisense gene. Transgenic plants expressing pTOM13 antisense RNA, accumulation of pTOM13 mRNA in wounded leaves was greatly reduced when compared with control and was almost undetectable in ripening fruit. In plants that inhereted two antisense gene segregated in a population of plants; ethylene reduction in ripening fruit was inhibited by 97%.
These fruit did not acquire normal redness and were much more resistant to shrivelling during prolonged storage. Another classic experiment is antisense RNA derived from expression of ACC2 cDNA of the LE-ACC2 gene result in 99.5% inhibits of ethylene in fruits, which fail to ripen when the level of ethylene was measured it showed below 0.1 ml of C2H2 per gram of fruit mass per hour. Antisense fruit kept in the air did not show respiratory burst even after 95 days.
A more refined investigation revealed that detaching the fruit from the plant even before ripening enhanced delayed ripening process. This unique interesting phenomenon was carefully noted in transgenic plants with reduced ethylene responsible for ripening behaviour of detached virsus attached fruit. That means transgenic plants attached fruit ripening at faster rate than detached fruit.
One of the possible reasons is due to the result of preventing transport of some unidentified factor into or out of the fruit. It has recently been shown that unidentified factor is probably a cytokinin which is exported from tomato fruit during ripening and has been implicated as an inhibitor of ripening. In addition, detached fruit exhibits diffusion of 97% of ethylene through stem scar with half-life of only 15 min.
On the contrary there is no stem scar in attached fruit, ethylene accumulate to higher level in fruit. Advantages of ACC synthase system over ACC oxidase is probably that it is less stable then ACC oxidase. The ACC oxidase system might be more susceptible to antisense inhibition. In addition, ACC synthase inhibition is carried out by 10 copies of ACC synthase antisense gene at one locus.
Expression of SAM Hydrolase:
This method never concern antisense technology but other strategies involved in the reduction of ethylene by manipulative process known as “metabolic interference”. When a substrate in a metabolic pathway that will normally be used to synthesize a specific compound is diverted to produce another non-toxic compound in the cell. A small company known as Agritope adopted metabolic interference method by expressing bacterial, viral gene encodes SAM hydrolase in transgenic plants.
This enzyme converts SAM a precursor for ACC to non-toxic byproduct that is recycled within plant cells. But most interesting point is that SAM is not used in synthesis of ACC and inturn reduction of ethylene in the plant. The gene from bacterial virus is modified to express SAM hydrolase in fruit specific at particular period of time specifically to mature green fruit before or just as it would normally start to ripen to block ethylene synthesis in fruit.
Expression of ACC Deaminase:
Another example of metabolic interference for ethylene reduction was developed by US based Monsanto Company. In this method, instead of directing SAM for the production of by product prevents ACC from being converted to ethylene by ACC synthase. Expression of an enzyme called ACC deaminase derived from bacterial metabolic ACC and ethylene production is drastically reduced.
Expressing ACC deaminase affecting ethylene synthesis has been examined. Evidence of direct correlation between degree of ethylene inhibition and rate of ripening was recorded when fruits are removed from the wine, the internal content of ethylene decrease rapidly.
As a consequence fruits are exposed to less total ethylene during the course of ripening and transgenic fruit ripen less rapidly. On the contrary, faster the rate of ripening of attached fruit has also been observing even in that case of plants transformed with ACC deaminase as it was in antisense ACC oxidase.
Antisense E8:
An additional example of another antisense transgene which affect ethylene production is E8 encodes for dioxygenase. This ripening related cDNA E8 has newly 34% amino acid sequence homology with ACC oxidase. Transgenic plants expressing antisense E8 transgene accumulates less than 10% of normal level of E8 protein. However, higher level of ethylene production was noticed due to unknown reason.