In 1729, French astronomer Jean Jacques d’Ortous de Mairan discovered a major biological event. Next to the instrument he was using to observe the skies, he kept a vase with the Mimosa pudica plant, a popular sensitive plant or creeper, whose leaves fold inward or droop when someone touches them. De Marian noticed that it was not always necessary to stroke the leaves to make them droop – at night, the leaves folded inward naturally and opened up again when the day dawned. Out of curiosity, he placed the plant in a closed chest which he kept in a dark cellar. To his surprise, even though there was no light, the plant’s leaves still folded and opened as if they had retained in their memory the length of day and night. One and a half centuries later, German botanist Wilhelm Pfeffer concluded that the movements of the Mimosa pudica in the dark originated from the plant’s internal mechanism: the so-called biological clock, a group of genes and other molecules that regulate the rhythm of physical and chemical phenomena – as exemplified by the movement of leaves, the blossoming of flowers, or the production of sugars (photosynthesis) – and maintains them in synchrony with the length of the day or with season changes.
Centuries after these initial experiments, a number of recent studies conducted at England’s Cambridge University with the participation of a Brazilian researcher, shed new light on how a plant’s biological clock is composed and how it functions.
Until some time ago, the general opinion was that the functioning of the biological clock was regulated by a group of ten genes and the proteins produced by these genes. Experiments led by Alex Webb, from the Plants Sciences Department at Cambridge University, showed that this was not true. The group, which Brazilian biologist Carlos Hotta is a member of, actually discovered that plants’ biological clocks are adjusted by much smaller molecules, such as cyclic adenosine diphosphate ribose (ADPRc), known for signaling extreme environmental situations to plants, such as water scarcity, lack of or excess sunlight, lack of soil nutrients or extreme cold or warmth.
“We already knew that the ADPRc was responsible for activating part of the plant’s protection mechanisms, among which are the closing of the tiny pores on the leaves to avoid water loss,” says Hotta, who played a leading role in the planning, development and analysis of the findings of the research study conducted during his doctorate studies at Cambridge from 2003 to 2007. “Now we have noticed that the ADPRc is also able to incorporate information on environmental changes into the plants’ biological clock, which regulates the plants’ physiology,” states the biologist, who is working on his post-doctorate studies at the Chemistry Institute of the University of São Paulo/IQ-USP. He is one of the authors that described the finding in an article published in Science last December.
This research study significantly changes the understanding on how biological clocks work; with possible implications even in agriculture. “We showed that part of the time-keeping mechanism depends on small molecules such as the ADPRc and not only on genes or proteins,” says Hotta. “This is a slight change in the paradigm.” In the past, researchers paid attention only to the genes’ activity levels; from now onwards, they will also have to learn how molecules behave inside plant cells and how they help adjust the biological clock.
The participation of the ADPRc as part of this time measuring mechanism allows scientists to understand, for example, why plants adapt so quickly to environmental changes such as changes in temperature or sunlight variations. Because it is so small, cells produce ADPRc in a matter of minutes, while the production of a protein, which is thousands of times bigger, takes up many hours. “This molecule apparently acts on the fine tuning of the biological clock,” says Hotta.
It was already known that in mammals the ADPRc is linked to cell organelle channels that store calcium and opens these channels. Like an electric switch, the released calcium activates and silences a series of proteins, acting like a chemical messenger. Evidence suggests that this mechanism functions in the same way in plants, controlling the opening and closing of the pores (stoma) of the leaves, the growth of the root hairs and the fertilization of flowers.
Hotta began his doctorate studies to develop his interest in investigating the function of calcium – and not of the ADPRc – in plant cells. “My objective was to discover if this chemical element influenced the functioning of the biological clock,” he explains. Previous studies had shown that calcium levels in plant cells varied during the day, increasing in daylight and decreasing in the dark, following a pattern that repeats itself every 24 hours – which is why this rhythm is referred to as circadian rhythm; that is, how it fluctuates during approximately one day. But the effect provoked by this oscillation was unknown. “Until some time ago, the general belief was that the biological clock sent information to the cells, using calcium as the messenger,” says the biologist. To the team’s surprise, the experiments revealed that the function of calcium is not to regulate photosynthesis and other processes. This chemical element is actually part of the biological clock itself, as if it were a gear in the center of this time-keeping mechanism. “Feedback occurs in this process; that is, the ADPRc controls the clock and at the same time is controlled by the clock,” says Hotta.
To come to this conclusion, the researchers used drugs that blocked the production of ADPRc in the Arabidopsis thaliana, or thale cress, used as a model organism to study several biological phenomena. The absence of ADPRc delayed the time-keeping mechanism. The cycles of movement in the leaves, the use of sugars to produce energy, or the opening and closing of the stoma, which had previously occurred every 24 hours, began to last for up to 27 hours. “All the clock-dependent rhythms that we measured became slower,” states Hotta. “This helped us conclude that the ADPRc is part of this time-measuring system that helps optimize plant growth.”
The speedy adjustment of the system allows the plant to prepare in advance for changes in the environment and be ready, for example, to capture carbon gas and begin photosynthesis before dawn, instead of triggering the process only after exposure to the first rays of sunlight. This same mechanism makes it possible for plants to produce molecules protecting leaves from ultraviolet radiation before the sunlight becomes more powerful in the middle of the day.
As the ADPRc adjusts what biologists refer to as clock time – the time that it takes a phenomenon to repeat itself – scientists believe that this molecule influences all biological rhythms controlled by the plant’s clock, such as for example flowering, photosynthesis, and the synthesis and breakdown of starch.
Such a huge influence has encouraged researchers to seek strategies to adjust the clocks of plants used in agriculture and to increase productivity. Although the study was focused on the Arabidopsis thaliana, Hotta believes that many of the findings probably hold true for other species. “Research work with other plants has revealed that various of the clock’s components are the same,” he says.
In another set of experiments with the Arabidopsis thaliana, Hotta noticed that the oscillation of calcium levels is controlled by the TOC1 gene (the acronym for Timing of Cab Expression of the protein regulator that binds to A and BL chlorophylls). A specific alteration – TOC1-2 – in this gene reduced the oscillation period of calcium levels and other rhythms to 21 hours. Changes in other regions of the gene caused such biological rhythms as the opening of the stoma and the movements of the leaves to last for 21 hours, while the calcium level oscillation lasted for 24 hours, according to a study published in Plant Cell last November. “This is an indication that a plant has two types of clocks, both of which have different characteristics, but depend on the TOC1,” says Hotta. Hotta is currently investigating the existence and functioning of biological clocks in sugar cane as the topic of his FAPESP-funded post-doctorate studies at the Cell Signaling Laboratory at IQ-USP.
The first step is to verify whether the biological clock of sugar cane is similar to that of the Arabidopsis and then investigate its role in the control of such characteristics as excess sugar and drought resistance. In the future, this information can lead to improvements and an increase in the productivity of sugar cane.
DODD, A.N. et al. The Arabidopsis circadian clock incorporates a cADPR-based feedback loop. Science. v. 318, p. 1789-1792. 14 dez. 2007.
XU, X.; HOTTA, C.T. et al. Distinct light and clock modulation of cytosolic free Ca2+ oscillations and rhythmic chlorophyll A/B binding protein2 promoter activity in Arabidopsis. The Plant Cell. v. 19, p. 3474-3490. Nov. 2007.