Questioning pre-established concepts, the medical doctor Anibal Vercesi, of the State University of Campinas (Unicamp), probed deeply into biochemistry and discovered in plants a type of protein that was believed to belong only to animals. His work could open up the door to the growing of transgenic tropical plants which are resistant to the cold, since, in animals, the uncoupling protein UcP is linked to hibernation, the lethargic sleep in which they survive at very low temperatures. It is because of UcP that the polar bear manages to maintain its body close to 37 degrees Celsius (°C) while in its state of hibernation. Rats kept for one or two weeks at temperatures between 5°C and 10°C also increased the production of this protein.
The UcP is produced in the fatty brown tissue, a fat that the mammals have when born on the back of their neck and which, in the majority of cases, including that of humans, disappears little by little. In the decade of the 70s, it was discovered that the function of the UcP is to produce heat (thermogenesis) and it was concluded that it had been an evolutionary acquisition of the mammals as an adaptation against the cold.
At the beginning of the 90s, while studying respiration and the conversion of energy in vegetable cells, Dr. Vercesi found similarities in the respiration cells of the potato and of the fatty brown cells of mammals. “As the potato originally came from the Andes, and was therefore adapted to the cold, I imagined that it was a type of polar bear of the vegetables”, reports the researcher. His finding contradicted well-founded understanding, but Dr. Vercesi confirmed that the vegetable contained a protein equal to that of UcP. Afterwards the Unicamp group identified the protein in tomatoes, corn, banana, mango, pineapple, peach, orange, melon, apple, avocado pear and strawberries.
With the collaboration of Hernan Chaimovich and Iolanda Cuccovia, of the Chemical Institute of the University of São Paulo (IQ-USP), the vegetable protein was tested in vitro and worked in a similar manner to UcP. The team decided to send its work to the magazine Nature which dedicated a page to it in 1995, but the skepticism was widespread. “In the plant physiology conferences, I was invited to participate mainly in order to receive criticism”, recalls Vercesi.
Finally, with the isolation in plants of the genes that codify the uncoupling protein, carriedout by the Molecular Biology and Genetic Engineering Center (CBMEG) of Unicamp, the theory was confirmed. From that moment until today, Japanese researchers have identified the protein in rice; Germans have found it in flour and potatoes, and the Australians in fruit. Today, the genome sequencing of vegetables has re-enforced the existence of the protein called Pump, the acronym for the plant uncoupling mitochondrial protein. It is uncoupling because of its capacity to dissociate two cellular processes, respiration and oxidative phosphorylation, which is the formation of molecules of ATP (adenosine tri-phosphate), form of cell chemical energy. The ATP comes from oxidation, a process that occurs within the mitochondria, a cellular organism which functions as a energy converter station.
A potential application of Pump is in the control of the ripening of fruit, which would reduce losses. Another is the production of genetically altered plants to resist low temperatures. The team from CBMEG has already managed to produce transgenic varieties of tobacco(Nicotiana tabacum) which super express with the Pump and grow more rapidly than the normal plants.
Dr. Vercesi is studying the role of the Pump in fruit such as the tomato, which could easily illustrate by its color the stages of ripening. In each step the Pump was isolated and it was proven that its activity increased as the tomato ripened. “It would appear that the explosion in the consumption of oxygen that occurs in the phase of ripening coincides with the increase in the level of the protein”, he states.
This study showed that, in the same way as the UcP in animals, the Pump needs free fatty acids in order to be activated. While the animal is hibernating, as well as providing substrates for respiration, the fatty acids activate the UcP. “It is for this reason that the bear fattens itself during spring and summer”, says Vercesi. “It needs a reserve of fat that will be consumed to generate heat during the winter.” With the tomato, things weren’t different.
When bovine albumin serum (BSA) was added to reduce the stock of free fatty acids, the albumin worked as an inhibitor of the Pump. The group discovered different forms of the Pump, in the same way that five variations of animal UcP have been identified. The UcP1 and UcP2 were found in white fatty tissue, the UcP3 in muscular tissue and the UcP 4 and UcP 5 in the brain. Instead of unleashing the stored heat, these variations had regulating roles.
Fat and thin
“The UcP2 of the white fatty tissue explains why two people with the same calorific intake put on weight in very different proportions”, comments Vercesi. “The person who puts on less weight registers a greater amount of the protein.”By way of comparisons, made easier by the sequencing of vegetable genomes, one now knows that at least four different types of Pump can exist. The Unicamp team discovered three of them in the sugar cane, one of them very similar to the UcP4 of the brain, and which appeared in different quantities in the organs of the sugar cane as well as at the distinct ages of each organ, a sign that they exert important roles in development. “The studies show that the Pump is not functionally similar to the UcP1, responsible for heat generation”, relates Vercesi. “The Pump would seem to have a greater similarity to varieties 2 and 4 and its prime function would appear to be to promote a regulation of the energy metabolism.”
In animals, the UcP1, activated by the cold, turns cellular respiration up to 50 times faster and promotes an accelerated burning of fat. On the other hand, the Pumps may not have such high activity. “Potatoes maintained in cold storage showed an increase in cellular respiration without a significant increase in temperature”, relates the researcher. One of the hypotheses is that the Pump stimulates respiration, decreasing the concentration of oxygen, and therefore promoting a defense against oxidation stress.
The Unicamp team tested the resistance to oxidation stress in tobacco plants, genetically altered, that clearly expressed the Pump. Normal and GM leaves were exposed to different concentrations of hydrogen peroxide (H2O2) – oxygenated water. The GM leaves proved to be more resistant to this type of aggression since they have lower levels of degradation of their chlorophyll and consequently maintained their color for a longer period of time.
The organisms only survive when they overcame the fight against oxidation stress. One of the main functions of fruit is to allow the transmission of the genome to the next generation, but the DNA, deoxyribonucleic acid, which carries the genetic code, is sensitive to oxidation stress. “During ripening and senescence, the uncoupling protein must be important in impeding the formation of free radicals and in preserving intact the genetic information”, says Vercesi. This would be one of the cellular defense mechanisms against substances such as calcium that stimulate the generation of species reactive towards oxygen. Such anti-oxidant potential could be manipulated to transfer the uncoupling proteins into natural food preservatives or future agents for combating degenerate illnesses.
The Metabolism of Oxygen and Calcium and their Relations with the Life and Death of Cells (nº 98/13012-5); Modality Thematic Project; Coordinator Dr. Aníbal Eugênio Vercesi – Medical Science School of Unicamp; Investment R$ 217,377.05 and US$ 366,158.88