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Secrets of sweetness

Study intends to identify the genes responsible for the sucrose in sugarcane

In the next three tear, special glass slides the size of a finger, on which thousands of genes or DNA fragments are inserted, may be able to reveal to man the paths of molecular biology that lead to the production of sweeter varieties of sugarcane. By 2007, with the help of the slides, called microarrays or DNA chips, São Paulo researchers intend to identify genes that favor the plant to accumulate high levels of sucrose, the popular cane sugar, during its process of maturing.

“If we discover good molecular markers involved in this process, we shall be able to develop more quickly genetically modified varieties that are richer n sugar”, says Glaucia Mendes Souza, from the Chemistry Institute of the University of São Paulo (IQ/USP), the coordinator of the studies. “In a pilot study, we identified 20 markers of the accumulation of sucrose, but the target is to find many others.” The microarrays will also be used to look for genes involved in resisting to pests (insects), in tolerance to the lack of water and in the interaction of sugarcane with natural bacteria that help fix nitrogen in their roots and work as fertilizers of the crops.

Plants like that, rich in sucrose and stronger, would form the ideal crop for the sugar and alcohol producers. That is why the project is receiving funds from FAPESP’s Partnership for Technological Innovation (PITE) program, from the Sugarcane Technology Center (CTC), of Piracicaba, controlled by the State of São Paulo Sugarcane, Sugar and Alcohol Producers’ Cooperative (Copersucar), and from the Centralcool sugar mill, from the town of Lucélia, in northwestern São Paulo. “We are investing in the improvement of our raw material, sugarcane”, says Carlos Yokio Nomura, Centralcool’s manager of logistics.

The expression of genes in 12 types of sugarcane, all of them developed by technicians from the CTC, by means of the traditional crossbreeding of plants with different characteristics, will be the target of an analysis by Glaucia’s team. “They are varieties that were created in our genetic improvement program, but they are not transgenic plants”, states agronomist engineer Eugênio César Ulian, the CTC’s manager for biotechnology. Producing transgenic sugarcane is a target for a second moment of the work, when the main genes responsible for the production of sucrose in the plant are identified.

If the project spawns technological innovations of a commercial interest, the three sources of funds will own the patents. Also, the universities of the researchers involved in the venture- besides USP, there are scientists from the State University of Campinas (Unicamp) and from the São Paulo State University (Unesp) engaged in the studies – will have a right to a portion of the royalties generated by possible patents.

Less concerned with producing basic knowledge for the academic world and more focused on finding technological solutions capable of increasing the competitiveness of sectors of the economy of São Paulo and of the nation, the PITE that is joining together São Paulo universities and companies from the sugar and alcohol sector is a direct offspring of a great scientific enterprise about sugarcane, the Sucest project, whose name stands for Sugar Cane EST, also known as Sugarcane Genome.

Prominent amongst the contributions from Sucest, which mobilized 240 researchers and was concluded in 2003, is the setting up of a large database of the plants genetic material. In it there is information about the sequencing of about 240,000 fragments of genes, called ESTs, or expressed sequence tags. “Sucest gave us the base for us to set up our project”, comments Glaucia, who is going to store the information about the functions of the genes discovered by microarray studies in a second, recently-constructed, database.

The DNA chips technology makes it possible to analyze which genes or pieces of genes of an organism are expressed (used) in different situations. It is not a tool for discovering genes that are difficult to identify, but rather one for mapping the role, the function of previously known genes or stretches of DNA, whose sequence had been determined by other techniques of molecular biology.

The methodology is much employed nowadays in work on the genetic bases of human diseases. In studies about cancer, for example, the microarrays are used for comparing the functioning of sets of genes in health and sick tissues. Accordingly, the scientists identify which genes are more, less and equally expressed by normal cells and by those with tumors. The experiments with DNA chips generate figures in which the genes inserted in the slides are represented by dots. The red dots represent genes that are more expressed in a given situation than in another. The green ones portray the less expressed, and the yellow ones those most used in both situations. If a gene proves to be overexpressed in a given context, such as in a tissue with cancer, it must be important for the occurrence of this condition.

Genomic fraction
In search of the molecular secrets that make sugarcane sweeter and more resistant to pests, the researchers are going to construct a microarray with 4,608 genes. This is not the total number of genes that make up the plant’s genome, but only a fraction of them – the most important fraction for comparative studies to be carried out. “Our preliminary results with a pilot chip with 1,920 genes were promising”, Glaucia reckons.  In the more crucial experiments of the project, which are going to try to discover the genetic bases of the early build-up of sucrose in the plant’s culm, the functioning of this set of genes will be measured in four kinds of sugarcane.

Two varieties produce a lot of sugar precociously, right at the beginning of the harvest, in the month of May. The other two varieties take more time to attain high levels of sucrose. “We are going to observe the expression of the genes during the plant’s whole process of maturing”, Ulian says. The comparisons will be made at four moments of the year: before the sugarcane harvest (in March) and at the beginning (May), middle (July) and end (September) of the harvest. The behavior of the genes will be analyzed in two kinds of tissues, in the leaves and in the culm (stem), where the sucrose is concentrated.

The studies that aim at finding genes for promoting other economically desirable characteristics in sugarcane will have a similar design. The molecular bases for the tolerance of the lack of water will be the target of work that is going to compare the expression of genes in four varieties of sugarcane, two adapted to dry climates and two not accustomed to aridness. Following this logic, of comparing the functioning of the same genes in varieties of plants with contrasting traits, varieties little and very resistant to diseases and susceptible to the action of nitrogen fixing bacteria will also be analyzed.

If everything works out, the researchers and the sugar and alcohol companies who are taking part in PITE will have identified genes capable of increasing the productive capacity of a sugarcane plantation. With this data, it will be possible, in theory, to produce genetically modified plants that are more efficient and safe for the environment – or, if the companies prefer not to create transgenic plants, getting new varieties by the classic process of genetic improvement.

The Project
Transcriptome of sugarcane (nº 03/07244-0); Modality Partnership for Technological Innovation (PITE) program; Coordinator Glaucia Mendes Souza – Chemistry Institute/USP; Investment R$ 555,693.00 – US$ 82,867 (FAPESP) and R$ 800,000.00 (CTC and Centralcool)