The red leaves of the small flowers of the Parrot’s Beak draw the attention of birds and pollinating insects. But many researchers are increasingly convinced that, in addition to transmitting messages to animals, the pigments responsible for most blues and reds of the plant kingdom, substances known as anthocyanins, fulfill vital functions for plant organisms.
A collaboration between laboratories led by two chemists, Frank Herbert Quina of the University of São Paulo (USP) and Antonio Maçanita of the Instituto Superior Técnico (IST) in Lisbon, Portugal, has been accumulating evidence that supports an old theory about anthocyanins: that these pigments protect leaves, especially the younger and older ones, against the harmful effects of excessive sunlight and its ultraviolet radiation.
“Anthocyanins have all the properties of a solar filter,” says Quina. In a series of articles, the most recent of which was published in March 2012 in Chemistry: A European Journal, Quina and his colleagues demonstrate how anthocyanin molecules absorb light and ultraviolet rays to quickly transform their energy into harmless heat for the plant.
The idea that anthocyanins protect leaves from the sun has been discussed since the 19th century. There was some doubt about this protective effect, because the anthocyanins were found in plant cells inside sacs called vacuoles, which at the time were regarded as mere cellular refuse. The situation changed in the early 1990s, when plant experiments showed that the photosynthesis of reddish leaves was more resistant to excessive solar radiation.
Light is essential for photosynthesis, which is the process by which energy from solar radiation is absorbed by a green pigment called chlorophyll and converted by a set of complicated biochemical machinery into sugars stored for later plant food. Excessive light, however, such as ultraviolet radiation energy, may overload and damage the chlorophyll, an effect called photoinhibition.
Newly sprouted leaves whose photosynthetic apparatus is not yet fully formed, are especially vulnerable to photoinhibition. “In the leaves of the cocoa tree, red when new, the anthocyanin starts to disappear as chlorophyll synthesis begins,” explains Quina, an American who has lived in Brazil since 1975.
The risk of photoinhibition is also higher for dying leaves. In the autumn in temperate countries, in some species of trees such as the red maple, the cells of the upper layer of leaves increase anthocyanin synthesis as the chlorophyll cells of the lower layer begin to break down in order to reuse the nitrogen of these molecules, which is stored for the winter.
Isolated in the laboratory, anthocyanins are red when placed in an acidic solution and blue in a basic solution. To study their reactions to light, researchers shoot laser pulses of varying wavelengths into anthocyanin solutions with different acidity levels in order to observe how they absorb radiation.
Red anthocyanin typically behaves as a weak acid, such as the acetic acid of vinegar. But when hit by a laser pulse, molecules become energized by light and are transformed into an acid as strong as hydrochloric by losing a hydrogen ion to water.
Everything happens in even less than the blink of an eye — in less than 200 trillionths of a second (picoseconds). This proton movement converts the energy of visible light and ultraviolet radiation into heat, and the molecules return to their weak acid form. “It”s a very efficient way to transform light energy into heat,” says Quina.
The proton movement, however, does not by itself explain the solar filter action. Indeed the process does not absorb enough ultraviolet radiation to protect the plant. What helps is the fact that the vacuoles are filled with colorless compounds known as copigments, which aggressively absorb ultraviolet radiation. Unlike anthocyanins, the copigments do not have mechanisms to dissipate the light energy without causing chemical reactions that damage the cell.
The concentration of anthocyanins and copigments in the vacuoles is such that the two classes of molecules when combined form a complex — a kind of super molecule — with the best protective properties of both.
Recent experiments by the Brazilian and Portuguese teams have demonstrated that the complex formed by one of the most common anthocyanins, cyanidin, and a copigment, coumaric acid, does not only originate from the copigment’s ability to repel water around the anthocyanin and thus draw near to it. There is also an electrical attraction between the positively charged anthocyanin and the negatively charged copigment, which causes the two molecules to forcefully stick together.
Thus, when ultraviolet rays are absorbed by the copigment, two separate processes may occur. If the anthocyanin is next to it, the copigment transfers light energy to it, which converts it into heat through the movement of hydrogen atoms. But if the molecules are stacked on top of each other, the light energy is transferred to the electron movement between them. This process occurs more quickly than the first — in less than one picosecond — and converts ultraviolet light into heat in an even more efficient way.
The solar filter action is not the only protection that anthocyanins offer plants. As with animals, plant metabolism produces free radicals — compounds rich in highly reactive oxygen that damage cells. Experiments by Quina and other researchers have confirmed that these molecules are potent antioxidants that quickly neutralize radicals. Incidentally, this is one of the reasons that nutritionists recommend a diet rich in vegetables such as purple cabbage and fruits such as grapes and açaí, all rich in anthocyanins.
Plants also have other antioxidant pigments. The most common are the carotenoids, which work with chlorophyll in photosynthesis, and are responsible for the yellow leaves in autumn and the color of carrots, tomatoes and annatto (achiote) seeds. Only one order of plants, the Caryophyllales, which includes beets, cacti and bougainvillea, produces other antioxidant pigments instead of the anthocyanins, the betalains.
No other plant pigment, however, gives rise to such a large variety of blue and red shades as do the anthocyanins. Their colors depend on the amount of acidity and the presence of certain copigments and metals in the vacuoles. That is why a single anthocyanin, a cyanine, under different conditions, is able to make cornflowers blue and roses red.
Mixed with a low concentration of copigments, anthocyanins change color easily with small alterations in acidity, which in most cases prevents their use by the food industry. One exception is the color of red wine, which occurs due to a reaction between anthocyanins. “We would like to find a way to stabilize the color of a pure anthocyanin,” says Quina. “For now we only know how to do this with inedible things, such as some detergents.”
Another problem in working with anthocyanins is obtaining them in large quantities. “It takes 20 kilograms of flowers from a relative of the sweet potato to extract 20 milligrams of anthocyanins from the flower,” says Amauri Marcato, a chemist with a PhD in botany and one of Quina’s collaborators at USP. In addition, obtaining a purified extract from the natural mixture of anthocyanins containing a single type of molecule is expensive and laborious.
Because of this limitation, the study of anthocyanins is usually done using other molecules as a model, the so-called flavylium salts, which are easier to obtain. Less complex than anthocyanins, flavylium salts have the same light-absorbing atomic structure as anthocyanins.
Quina and Marcato hope to work around these problems in the near future by trying to produce anthocyanins in vitro. The idea is to culture in the laboratory a liquid with undifferentiated plant cells, originating from a young plant. Controlling the conditions of the culture, such as light, they hope to induce some of its cells to produce anthocyanins in large quantities. “It would be much easier to separate them,” says Marcato.
Quina’s plans also include collaborating with molecular biologists to manipulate the synthesis of anthocyanins. He believes that a variety of cocoa whose leaves grow red throughout their lives will soon be produced. So the tree would be able to grow in the sun, and it would help get rid of witch’s broom fungus, the principal pest of cocoa.
FERREIRA DA SILVA, P. et al. Photoprotection and the photophysics of acylated anthocyanins. Chemistry: A European Journal, v. 18. 2012.