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Genomics

The genes of paper

Teams from five states identify the DNA sequencing that could turn the eucalyptus more resistant to drought and diseases

Eduardo CesarOne of the goals: to control the active genes in leaves in order to increase defense against fungi and insectsEduardo Cesar

Over the next few months in the laboratory of agronomist Ivan de Godoy Maia, in Botucatu, interior of Sao Paulo State, the first shoots of tobacco with a special characteristic should sprout up: genes that are not originally of this plant of light green large leaves, broad and soft, but from the eucalyptus, a tree of 35 meters in height and hard leaves in the form of a lance. Since the end of 2005 Maia has been working on the preparation of six genes of this tree, apparently active in a single vegetable tissue – the leaf, the stalk, the root, the flower or the fruit – in order to implant them into tobacco. The reason for producing shoots of transgenic tobacco is that this plant grows and reaches its reproductive phase in around six months, at least twelve times quicker than that of the eucalyptus. Thus, it is possible to confirm in a short period of time in which vegetable  tissue each one of these genes acts and its probable function. To control this and other genes is the first step towards creating eucalyptus trees that are more resistant to drought and to pests or that are capable of producing better quality wood for the extraction of cellulose and the production of paper.

These experiments with tobacco or with another model plant, the Arabidopsis thaliana, are those that the geneticists call the functional genome – in this case, it is the third stage of the Eucalyptus Genome Project, the first tree genome sequenced in Brazil. It is hoped that within a few years the results obtained in the laboratory create more productive plantation of eucalyptus. A native plant of Oceania, this tree was introduced into the country at the start of the 20th century by Edmundo Navarro de Andrade in order to produce sleepers for the railway lines that advances into the interior of the state of São Paulo. Brazil possesses the largest planted area in the world destined towards commercial ends. There are 3.5 million hectares that guarantee to the country the position of the 7th world producer of cellulose and 11th in paper, activities responsible for 4% of the Gross National Production (GNP), or R$ 80 billion.

The search for genetically modified varieties of eucalyptus is the final part of a genes sequencing project that began back in 2001 and, with a profile that is uncommon in the country, brought together universities and companies. In the previous phase, called data mining, completed at the end of 2005, genes were identified that could contribute to the perfection of this tree. With the help of computer programs, teams from 20 universities from the States of Rio Grande do Norte, Alagoas, Pernambuco, Rio de Janeiro and Sao Paulo searched through 123,889 gene segments. This total corresponds to almost 15,000 of the genes that had been sequenced by the consortium Eucalyptus Genome (Forests), formed by FAPESP and four companies in the wood, paper and cellulose industry – namely, Duratex, Ripasa, Suzano and Votorantim – with the collaboration of teams from the Agronomy and Environmental Genomes (AEG) Project.

During the data mining the researchers compared the genes of five species of eucalyptus common to the country – Eucalyptus grandis, E. urophylla, E. camaldulensis, E. saligna and E. globulus – with already known genes of plants such as tobacco, poplar, and Arabidopsis. And they discovered almost 200 protein producer genes of the defense system against pathogens and 15 others responsible for a protein complex that silences other genes. Other results are related in the 19 articles of the magazine supplement Genetics and Molecular Biology of November 2005. “These and other genes must serve as indicators of the characteristics that are desired to be reproduced in the plants” states the forestry engineer Luis Eduardo Aranha Camargo, from the Luiz de Queiroz Upper School of Agriculture (Esalq) of the University of Sao Paulo (USP), the coordinator of the second and third phases of the Eucalyptus Genome Project.

Over the last forty years traditional genetic improvement techniques such as those used in the 19th century by the Austrian monk and agronomist Gregor Mendel, one of the founders of genetics, have made possible an increase of up to four times in the productivity of Brazilian eucalyptus. The forest’s wood production per hectare leaped from 12 cubic meters per year in 60s to as high as 50 cubic meters. This annual production corresponds to a room of 5 meters in length by 4 meters in width filled up to the ceiling with wood obtained by cutting down trees planted in an area equivalent to a block.

But not always does the simple crossing of distinctive varieties produce the desired effect. For this reason what is being attempted in producing genetically modified eucalyptus trees is to alter characteristics that are not easily manipulated by way of traditional genetic improvement. An example is the control of the concentration of lignin, a natural polymer that functions as a cement between cells and gives hardness to the wood. The dream of all paper and cellulose producers is to collect plants with a level of lignin lower than normal, namely that between 20% and 30% of the tree’s mass. Low levels of lignin would simplify the processing of the wood to obtain cellulose, the sugar formed by thousands of molecules of a simpler sugar, namely glucose. On the other hand for the furniture industry and the steel making industry, which uses timber as fuel , trees with higher levels of lignin are what interest them.

The control of the quantity of lignin will depend on the ability of the researchers to control one or more genes identified by the  Ricardo Harakava, from the Biology Institute of Sao Paulo. Harakava found 13 genes that regulate the production of lignin, previously known only in herbaceous plants, such as Arabidopsis thaliana, and in the poplar, a tree of up to 35 meters in height, the main source of cellulose in the northern hemisphere countries. The comparison of the level of activity of these genes in different species of eucalyptus – there are around 700 – could reveal a functional standard typical of trees with low lignin production, thus working as biological markers for these characteristics. “With the help of biological markers it’s possible to make a precocious selection of plants with a low level of lignin” exemplifies Harakava. Another possibility: to reduce the activity of these genes by way of transgenic techniques, still in the experimental phase in the case of the eucalyptus.

Natural antibiotics
It is also intended to develop eucalyptus trees that are more resistant to diseases. In the warmer and more humid regions of Brazil, an elevated variety of pathogens especially attack the young plants and impede their growth. Species such as the Eucaliyptus urophylla, widely used for its rapid growth rate and which reaches its reproductive age in only five years, is highly susceptible to fungi that digest the wood, damage the leaves or cause the rotting of its roots. By analyzing the data from the Eucalyptus Genome Project, the team led by the geneticist Ana Maria Benko-Iseppon, from the Federal University of Pernambuco (UFPE), located 210 genes capable of helping to solve these problems. “This is a very high number, since the genetic material was extracted from healthy plants” says Ana Maria. “This result suggests that there might be an even greater number of resistance genes in the eucalyptus.”

These 210 genes belong to the five resistance gene classes (R genes) known in plants and perhaps there might be even a new class, the sixth, which has still to be confirmed by way of experiments. Associated to different mechanisms of the eucalyptus’s defense system, they unchain chemical signals that induce a collective cellular suicide or stimulate the production of compounds that act as natural antibiotics and eliminate the fungi, bacteria, viruses or worms.

On the other hand , the work of the geneticist Márcio Alves Ferreira, from the Federal University of Rio de Janeiro (UFRJ), could help in the development of eucalyptus trees that are less sensitive to drought – lack of rain for more than a month is a threat to the plantations in the northwest region of  the state of São Paulo. The team from Rio have found 50 regulatory genes of the family known as homeobox, which switch on or switch off other genes related to the tolerance to lack of water. “If the experiments come out right, we could be producing varieties resistant to drought using genes of the eucalyptus itself and not of another species” explains Alves-Ferreira. His work with the genes MADS-box, responsible for the development of the flower, could impede the production of pollen grains and the contamination of normal plants with the genetic material of transgenic varieties.

Genetic Interrupter
The group led by the biologist Marcelo Menossi, from the State University of Campinas (Unicamp), have identified 8 very active genes in almost all of the tissues of the eucalyptus. These are genes such as that which contains the recipe for catalase, the antioxidant enzyme that protects the cells from damage caused by free radicals, and the one responsible for the production of the heat shock protein, HSP, which avoids cellular damage brought about by an increase in the environmental temperature. A further 13 genes distinguish themselves from the others as they function on a single tissue.

In order for the research to advance, it is essential to understand the tissue in which the genes act. Each gene contains an initial strip called the promoter region or regulator that acts as a genetic interrupter, controlling where the gene is going to function or shutting it off. In this manner, a gene that coordinates the production of flower petals during the reproductive cycle is going to manifest itself only in the floral tissues – and will remain inactive in the root or trunk – because its promoter region will become active only in the flower cells.

In the case where it is desired to protect the eucalyptus from insects that attack its leaves, it is possible to associate a gene that produces a toxin against the insect at a promoter region of an active gene only in the leaves. If it were the goal to generate a variety resistant to the fungi that attack all of the tissues, it would be necessary to associate a gene against the fungus to an active promoter throughout the plants organs. It was exactly the promoter region of six of these genes – two specific to the leaf, one to the root, one to the floral bud and another to the floral and fruit bud, as well as a gene active throughout all of the tissues – that Ivan de Godoy Maia has obtained in his laboratory at the São Paulo State University (Unesp), in Botucatu, and is now preparing to insert into tobacco plants. If things work out, the researchers will have at their disposal promoter sequences identified in Brazil and will no longer need to pay royalties for the use of promoter strips already patented abroad.

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