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The architects of the new sugarcane

The knowledge accumulated by the Sugarcane Genome researchers complements traditional genetic improvement techniques and shows how to develop more productive and pest-resistant varieties.

The ideal sugarcane is achievable. More productive, pest-resistant, tolerant of drought and weed killers, and efficient at absorbing nutrients to the point where it survives best in acid or not very fertile soil, this sugarcane may take shape based on the Sugarcane Genome discoveries, which is reaching its conclusion, a year and a half after having been publicly announced. The 240 researchers working on this project – the first plant sequencing to be carried out in Brazil – have identified around 80,000 genes giving a complete map of how the plant lives, reproduces, and dies – and, properly manipulated, they may make it possible to achieve these characteristics so often dreamed of by farmers and sugar and alcohol manufacturers.

The redesigned sugarcane already has a timetable for it appearance. “Within two years, the first varieties resistant to two pests, the bacterium Leifsonia xyli and the fungo-do-carvão (coal fungus) should be ready, at least in the laboratory”, announces Paulo Arruda, the DNA coordinator, in charge of 60 laboratories, 22 of which taking part in the sequencing and the other 48 are dedicated to mining or prospecting for data, the so-called data-mining. “We can look forward to greater things”, says Éder Giglioti, a researcher at the Federal University São Carlos (UFSCar). It is not yet a priority, but, in his opinion, we can already imagine using the sugarcane as a bioreactor, able to produce not just sugar and alcohol, but also chemical compounds of interest to the pharmaceutical industry, as it is already being done in other countries.

Soon, the Sugarcane Genome or Sucest, from Sugarcane EST, should open up into two paths. On the one hand, applied research should be strengthened, searching for new varieties of sugarcane. On the other, basic research continues, focusing on deeper understanding of the sugarcane’s biological mechanisms. Arruda believes that these two paths will cross and benefit from each other permanently. “We will join the frontier of scientific knowledge with the search for results, he says. The innovation is the participation of sugar and alcohol producers, with whom he has engaged in intensive talks in recent months. The objectives: to identify the specific problems to be worked on with the information culled from the Sugarcane Genome and to find new partners to finance part of the research in this new stage. “We want this work to help solve practical problems”, he says.

With this approach, the research of three genetic improvement laboratories, which already took part in the Genome project and have frequent contact with sugarcane growers, should be still further valued. They are: The Technology Cooperation Center of Sugar and Alcohol Producers in the State of São Paulo (Copersucar), which in this initial stage has contributed around US$ 400,000 to the project, the Campinas Agronomy Institute (IAC) and UFSCar’s Agrarian Sciences Center, at Araras, built using the premises of the National Sugarcane Improvement Plan (Planalsucar), abolished at the beginning of the 90s.

After totting everything up, the Sugarcane Genome should be completed almost a year ahead of schedule and cost only half the US$ 8 million approved by FAPESP. The saving comes, in part, from the use of already installed infrastructure and the team’s experience: of the 32 initial laboratories, 15 in the sequencing group and 8 employed in data mining, had already taken part in the Xylella Genome project, a pioneer project sequencing the Xylella fastidiosa bacterium, completed at the beginning of the year, which put the Brazilian scientific community in the front line of world genomics.

Replicas of the clones of the sugarcane, the Xylella, and in the future, two other bacteria the Xanthomonas citri and the Leifsonia, the mapping of which is currently underway, will be held at the Clones Storage and Distribution Laboratory, scheduled to begin operating next month on the campus of the São Paulo State University (Unesp) at Jaboticabal, and thus make Brazil a supplier of genetic material to the world (see box).

The researchers have exceeded the initial target of 50,000 active or expressed genes directly associated with the plant’s metabolism. The functions of two thirds of this total are already known, because of the similarity with the genes of other organisms, described in international databases. The other part is still more important: one third of the genes found are novel, with no counterpart in other organisms and there may lie the origin of the more peculiar characteristics of sugarcane. The researchers intend to organize this mountain of information by December in the form of a gene index, a list of sugarcane genes grouped by function. There are 40 structured categories and 15,000 genes have already been classified, among which are eight or ten associated with the production of sucrose, whose action can, in principle, be made easier in the pursuit of a sweeter sugarcane.

Comparisons
The work of analyzing the data, data mining, means spending hours in front of a computer searching for genetic similarities between sugarcane and other plants, and even other species, including microorganisms, animals and man. Authentic prospecting: there are around 500 sequencing projects in course around the world – from apples to the domestic cat. It was this way that, at Unesp’s Agronomic Sciences School, in Botucatu, Eiko Eurya Kuramae found 240 genes related to the production of substances that fight against pathogens (funguses, viruses, bacteria) and insects. With these, Eiko has built a model of how the plant reacts when faced with an external attack – strategic knowledge when trying to develop plants that are more pest-resistant. It is a difficult struggle swinging from side to side. When attacked,  the plant tries to stop the microorganisms from getting in. If it cannot do so, it produces toxic substances that inhibit the invaders’ progress (see illustration on page 32). The reaction depend on the interaction of the systems with the pathogen, the age, the tissue attacked and plant’s nutritional condition. “The genetic control of resistance to pathogens in plants is determined by the gene to gene interaction”, says Eiko. “The resistance response is induced only if the pathogen encodes a specific avirulence gene, the avr, and the plant carries the corresponding resistance gene, the R”. The disease only takes hold if the plant’s R gene and the invaders’ avr do not exist or are inactive.

Vicente Eugênio de Rosa Jr., under the co-advise of Eiko, is dedicating his doctoral thesis to one of these substances, jasmonic acid. Marleide de Andrade Lima is working on the other, salicylic acid, in her postdoctoral studies. These studies in this field have practical applications: the plant’s defensive system can be extended by expressing the genes associated with the production of these defensive substances.

Suzelei de Castro França, at the University of Ribeirão Preto (Unaerp) has also worked on the defensive substances. Studying the genes expressed in sugarcane, she discovered that each tissue of the sugarcane has different reactions to the pathogens, predators, or injuries in general, favoring different chemical compounds at one time or another, depending on the situation (see illustration on page 33). At present, Suzelei is engaged on the study of cell signaling, the mechanism whereby the different organs of sugarcane communicate with each other, and gradually create alternatives for manipulating the production of the substances associated with the stress. From the agronomic standpoint, this means healthier plants, resistant to inclement weather and, therefore, more profitable crops.

Essential functions
You get the false impression that the interior of a cell has no more secrets to reveal, such is the familiarity with which researchers speak of the intimacies of the sugarcane and add new items to established knowledge. An example is the mitochondrion, a compartment of the cell that has an already established task, the production of energy. But, Francisco Gorgônio da Nóbrega, of the University of the Vale do Paraíba (Univap), in São José dos Campos, saw that the mitochondrion fulfills other essential functions. One of them is the production of the so-called iron-sulfur center, associations of iron and sulfur atoms whose function is to transport electrons inside the cell. As he demonstrated, they also form as sort of box giving stability to the proteins manufactured by the cell.

Cellular reproduction also became reasonably clear. Paulo Ferreira and Adriana Hemerly, of the Federal University of Rio de Janeiro (UFRJ), one of the first data mining groups from outside São Paulo to join the project, in April last year, have studied it. They have discovered 15 exclusive sugarcane genes so far, associated with at least four different forms of a protein called kinase, whose function is now well known: they are the ones, according to Ferreira, that activate and deactivate each stage in the cellular cycle, from the duplication of the DNA to the separation of two cells “Kinases are triggering proteins that set off the cell division process and allow the action of the proteins of the origin recognition complex, connected to the DNA”, he says. There are also the proteins called anaphase promoter complex or APC, triggered by the kinases, which the cell afterwards destroys in order to allow the chromosomes to separate.

Not everything is understood, obviously. Carlos Martins Menck of the Biomedical Science Institute of the University of São Paulo (USP), is investigating how the organisms fix the DNA molecule – through other molecules. The repair proteins recognize the damage, assemble the enzymes that eliminate the injured section and pave the way for other enzymes that will put the section that should have been correct from the beginning in the DNA. If the DNA remains damaged, serious disease occurs, such as Cockayne’s syndrome, with development and mental retardation problems.

Menck compared the replication genes in humans, yeast (a unicellular organism), and two vegetables, sugarcane and Arabidopsis thaliana, a plant in the mustard family with only five pairs of chromosomes used as a model in molecular biology studies. He found 85 genes in common, a 73% similarity. More refined analyses suggested the proximity between the groups. “Our repair system is closer to that of a plant than it is to yeast”, he says. Of this total, one third of genes found in the sugarcane had not previously been described in plants.

The combinations are intriguing – and they suggest that, more than simple similarity, it seems there is a unity between living beings. According to Menck, there are repair genes in bacteria that apparently do not exist in humans, but they are present in plants. At the same time, genes important for repairing DNA, such as the one  known as XPA, present both in humans and in yeast, have still not been found in Arabidopsis and in sugarcane. Menck has a theory ”It is possible that plants have different or redundant DNA repair  mechanisms”. But there is still no way of understanding what the gene BRCA1, a deficiency of which causes breast cancer in humans, is doing in sugarcane.

Cause of mutation
The behavior of transposons – the jumping genes that leap from one chromosome to another, discovered in the 40s by the American geneticist Barbara McClintock (1902-1992, Nobel Prize for Medicine 1983) and accepted with considerable reluctance by the scientific community – is also not yet clear. Marie-Anne Van Sluys, at USP’s Biosciences Institute, did not expect to find many in sugarcane, but she found no fewer than 13 different types of transposons.

It used to be thought that the jumping genes functioned in very specific places, in the same tissue. But not so. As she discovered, more than one of these restless genes is active in cells in the same tissue at the same time. “The capacity of the transposons to be expressed together in the same tissue has never been assessed”, says Marie-Anne, who lets slips two questions. Why are there different transposons in the same tissue? Do they have any other function that we do not yet know about? In bacteria, they are associated with resistance to antibiotics. In drosophila, the fruit fly, they ensure the structure of the telomeres, the tips of the chromosomes. Because they jump about a lot, the transposons cause mutations and the genetic variability of the species, selected throughout the process of evolution. It is for this reason, that Marie-Anne sees them as candidates as genetic markers for the species of sugarcane to be developed.

Like rice, another plant in the gramineous grass family, sugarcane has at least one similar gene, the XA21, which gives it resistance against  the bacterium Xanthomonas orizae. From this, Luís Eduardo Aranha Camargo and the graduate student Mariana Sena Quirino, of USP’s Luiz de Queiroz Higher Agriculture School (Esalq), decided to work by trial and error: they want the XA21 to be even more efficient against a similar bacterium, typical of sugarcane, the Xanthomonas albilineans. They carried out experiments favoring the expression of this gene and now they are examining the DNA of the parent plants and the daughter plants.

The research showed four variations of the same gene (alleles), “perhaps with different functions”, says Carmargo. At this point, the work becomes more complicated. “We can use the information from the sugarcane genome to look for the ancestral genes, responsible for the modern sugarcane”. If all goes well, they will learn which alleles came from the Saccharum officinarum or from the Saccharum spontaneum, the two species that are the ancestors of modern sugarcane, an organism considered complex.

The sugarcane used today to produce sugar, alcohol, brandy, and the prosaic raw brown sugar has a variable number of chromosomes – from 100 to 130. One of the probable reasons is that in each cell is kept, at least in part, the genetic loads of the original species – the S. spontaneum has from 36 to 128 chromosomes, and the S. officinarum between 70 and 140. The present-day plant is a hybrid or, from the cellular viewpoint, a polyploid organism: each chromosome has from six to ten copies – not always precisely the same. This peculiarity caused the full sequencing of the genome to be ruled out from the beginning. It would probably be expensive, wearing, and take too long.

Special sugars
As an alternative, the Genome team worked by sampling, making use of the Expressed Sequence Tags technique (EST), which speeds up discoveries by identifying only the sections of the expressed genes, responsible for forming proteins. Discoveries were made at a commendable pace. In March 1998, at the time of preliminary talks about the project, the plant ESTs amounted to 4.8% of the total deposited in the Genbank, in which human ESTs came to 63.4%. Within two years, the number of plant ESTs had risen more than fifteenfold, and today they represent 18.2% of the total (see chart).

In the sugarcane expected based on the genes so far discovered, the sugar content is not a disturbing problem: since the 70s, the productivity of sugarcane has grown by 1% a year, through traditional genetic improvement, by crossing and selecting new strains. Brazilian sugarcane produces between 120 and 130 kilograms of sugar per ton, equivalent to international standards, although slightly lower than Australian ones (140 to 150 kilograms per ton).

The discovery that there are 162 genes associated with the sugar metabolism in general (44% of the genes already described in plants and animals with the same function) encourages setting higher goals, such as the prospect of producing special sugars. This is true of trealose, which, as well as having higher commercial values, has biological importance: in other organisms, it provides resistance to cold and dry conditions, a little-exploited feature in sugarcane.

Eugênio Ulian, a researcher at Copersucar who found two genes leading to the synthesis of trealose is not looking ahead only. In July this year, he reminds us, there was a severe frost in São Paulo, which adversely affected corn and coffee plantations and, to a lesser degree, sugarcane. Ulian believes that the losses could have been smaller if the mechanisms for activating the production of trealose had been clearer.

Nationwide scope
These prospects are evidenced based on the integrated work of the laboratories spread over the three public state universities (USP, Unicamp and Unesp), three private universities (in Ribeirão Preto, Mogi das Cruzes and São José dos Campos), in the IAC and in Copersucar’s Technology Center. They make up the ONSA (Organization for Nucleotides e Sequencing and Analysis) network, supported at two central points, the Molecular Biology and Genetic Engineering Center (CBMEG) and the Bio Information Technology Laboratory, both at Unicamp – the first preparing the material to be sequenced by the laboratories (it produced 1 million clones of the sugarcane genome) and the second organizing the information in the database, analyzed by the data mining groups.

Since the planning stage, the research has relied on the international support of the steering committee, the external evaluation  committee, formed by Jean Christophe Glaszmann, of the International Center for Cooperation in the Development of Agronomics Research (Cirad), of France, and Andrew Paterson, of the University of Georgia, in the United States. Although it is a São Paulo project, the results are increasingly shared by other states.

At the end of last year, two further groups joined, one from the Federal Rural University of Pernambuco (UFRPE) and the other from the Federal University of Alagoas (UFAL), enjoying the support of the respective state research foundations. In July, another move forward: another 36 data mining groups joined, including groups from research institutes in Minas Gerais, Paraná, Bahia, Rio Grande do Norte and Rio de Janeiro. And so large is the quantity of information produced that Paulo Arruda, the project’s coordinator, is encouraged with the possibility of giving room to new teams, as of next year.

“We need creativity and imagination to make use of this information in the best way possible”, says USP’s Menck. The work should pay off. In going beyond the limits of traditional genetic improvement, what we now know about the genome may shorten the development time of new strains of sugarcane. This is usually a lengthy process, taking from 12 to 15 years. “If we can cut the time by one year straight off, that’s fine, says William Burnquist, manager of the Copersucar Technology Center. The modest aspirations are deceptive: every year means investment of around US$ 8 million. When this target proves achievable, using genetic markers that help select sugarcane strains with the desired characteristics right at the beginning of this marathon, the saving achieved will be double that spent on the research so far.

Clones from Brazil to the world

Ready to be opened, the Brazilian Clone Collection Center (BCCC), in Jaboticabal, will keep the clones produced in genome projects under lock and key, in an air-conditioned room at 20 degrees centigrade, with a double polystyrene lined wall, inside eight freezers kept at 86 degrees centigrade below freezing point, under constant surveillance. When requests for clones are received, a system of robots equipped with a video camera, will collect the bacteria on the petri dish, and organize the samples on micro-dishes, with 96 or 384 holes, or on high-density membranes to be used in genetic manipulation experiments.

It is the first center of the sort in Latin America. Set up at a cost of US$ 240,000, it will work along similar lines to the American Type Collection Clones (ATCC) or the Image Consortium banks, in the United States. It will be able to serve public institutions at cost price, nowadays between US$ 30 and US$ 50, under  a written commitment that the material will be used solely for academic purposes, without commercial intent. “Within three years, the laboratory will have to be self-sufficient”, says Jesus Aparecido Ferro, one of the Sugarcane Genome coordinators who will be in charge of the new laboratory.

According to him, companies and private research institutions will be given differentiated treatment. “A supervising committee will examination whether the clone can or cannot be sold”, he says. “In an extreme case, the request may be turned down”. And, he adds, as a security measure, they will work only with the name of the gene and an indication of the homology (similarity) of the gene requested with those of other organisms. The sequence of the bases, essential to the study of genetic manipulation, will remain  confidential”, explains  Jesus Ferro.

Old source of wealth

Brought by the Portuguese from the island of Madeira, sugarcane arrived in Brazil in 1502. It has never ceased being a source of wealth for the country. The crop covers 5 million hectares and, at each harvest, Brazil produces 300 million tons of sugarcane, equivalent to 25% of world production, converted into 14.5 million tons of sugar and 15.3 billion liters of alcohol. It also involves 350 companies, around 50,000 producers and about 1.4 million direct workers and another 3.6 million indirect workers, according to Copersucar. The state of São Paulo is the biggest producer in Brazil: the crop turns over US$ 8 billion and directly employs 600,000 workers in the State.

The Sugarcane Genome was designed with a clear economic focus in mind, for the purpose of increasing the productivity of the sugar and alcohol industry. It also faces looming challenges. One of them is fighting the bacterium Leifsonia xyli subsp. Xyli, which causes one of the most serious sugarcane diseases in the world, ratoon stunting, which has caused losses estimated at US$ 2 billion to Brazil over the last 30 years.

And sometimes, new problems arise. Éder Giglioti, of UFSCar, characterized a new sugarcane disease, the false red wrinkles (falsa estrias vermelhas), so far found only in Brazil. In his opinion, there is some evidence to suggest that it is a new species of Xanthomonas, the fifth of the type to be isolated in sugarcane.

The project
Sugarcane Genome (97/13475-2); Type: Research project within the scope of the FAPESP Special Genome Project; Coordinator: Paulo Arruda – Molecular Biology and Genetic Engineering Center of Unicamp; Investment: US$ 4,484,090.61

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