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New model

Mutant tomato plants help unveil the genes and hormones that regulate the lives of plants

eduardo cesarThese tomatoes correspond to the widely-held image of a mutant: weird, to say the least. A spontaneous genetic alteration transformed the stamens – that fall off when the flower dries up – into female reproductive organs, the part from which the fruit comes from. The result is multiple tomatoes, like Siamese twins. Normally invisible to the untrained eye, most mutations are noticed by specialists, who found in the genes and hormones of the mutants a tool to understand the “private life” of plants. “We are only really able to understand the function of gene if we are familiar with the gene’s variations”, says Lázaro Peres, an agronomist from the Escola Superior de Agricultura Luiz de Queiroz (Esalq Agronomy College of the University of São Paulo/USP), located in the city of Piracicaba.

To understand how genes and hormones regulate plants, the researcher used a miniature tomato plant variety known as Micro-Tom. These are small plants with a short life span. Peres grows approximately 3 thousand tomato plants in the greenhouse. These tomato plants, which are only 8 centimeters tall and 70 days old, are top-heavy with fruit and accumulate mutations for all tastes. With the help of these mutants, the group from Esalq has been able to gain in-depth knowledge on how plants resist drought, perceive light and develop fruit with more concentrated soft mass.

In 2006, Peres discovered a gene that regulates the efficiency of how the plant uses water. He named this gene “well”. The traditional definition of a well is a shaft sunk into the earth to tap the underground water. It is also the name given to each hole in the trays where the tomato plants are grown; in addition, “well” is an acronym which stands for “water economy lycopersicon locus”. The mutation of the Well gene is a natural genetic variation that controls cellular differentiation and allows for survival under arid conditions. This mutation defines, for example, the proportion of cells that will turn into stomata, the respiratory and transpiration pores located on the surface of leaves. In addition to affecting the number of stomata, the Well mutation produces anatomic structures that allow the plant to produce more photosynthesis with less water. The project is headed by post-doctoral student Ricardo Fornazier, who is conducting the measurements to fill in the details on how this process works, which is useful to reduce the amount of water necessary to cultivate not only tomato plants but for agriculture in general.

Besides the mutations, kin species are also an important source of genetic variation. The team from Esalq looked for these variations in the Lycopersicon hirsutum, a close relative of the domestic tomato plant (L. esculentum). The hirsute leaves and fruit provide resistance to insects. In addition, this plant is accustomed to the cold weather in the Andes mountain range, as it is native to that region. By cross-breeding these two species, a technique that has been used ever since human beings became farmers, Peres was able to obtain cold-resistant micro tomato plants. “We are interested in studying the genetic variations of wild plants, because these variations are the result of survival of the fittest,” he explains. The success of the Micro-Tom as a plant model has placed the agronomist from Esalq in an outstanding position: “No one else in the world has a collection of mutants of the kind we have here.”

But genes are not enough to understand the physiology of plants. Peres explains that hormones are the elements responsible for shaping the majority of the characteristics and of the vital functions. Until recently, very little was known about these hormones – until researchers started using mutants. To study the plant hormones, he cultivates tomato plants with mutations that make these plants insensitive to the actions of those substances.

Beyond the genes
Some of Esalq’s mutants help understand how plants perceive light. When the sun is out, the plant gets the message that this is the right time to produce chlorophyll for photosynthesis, the process that uses sunlight to convert carbon dioxide into sugars, the plants’ energy reserve. The elements responsible for this plant vision are pigments known as phytochromes. A mutant with phytochrome deficiency does not perceive the light well enough and even if it is exposed to full sunlight, it turns yellow and grows long branches, as if it were not getting enough light. Doctoral student Rogério Carvalho was able to generate double-mutants, which combine the mutation of the phytochrome with other mutations that render the plant insensitive to specific hormones, to investigate which of these hormones are used by the phytochrome to shape the plant?s response to light. He discovered that a high response to light involves a low concentration of gibberellins and a high concentration of cytokinin. Peres explains that this discovery has a direct application, as the level of these hormones can be manipulated by adding chemical substances to the plant.

Hormones are also used in the production of seedlings from stakes. The researcher applied the auxin and cytokinin hormones to each species by using the trial and error method, until he achieved a combination that induces a piece of the plant to produce roots and stems. To make this process less empiric, the researcher sought to understand how the   plant works, which is the capacity of the cells to turn into any kind of tissue. Unlike animal stem cells, which concentrate their versatility on the embryonic stage, plants need competent cells during their entire life span – to be able to produce roots, leaves, fruits, flowers and stems at any moment.

eduardo cesarCross-breeding with wild species generates cold-resistant tomatoeduardo cesar

At the Esalq laboratory, students lined up bits of leaves on plates with nutritious gelatin: from the bits of leaves taken from the mutants to extract the Rg1 gene, a name derived from regeneration, roots sprouted from the edge of the leaf fragment. The Rg1 gene was discovered by a Dutch researcher in a wild relative of the tomato plant; this researcher then distributed seeds all over the world. Peres transferred the gene to the Micro-Tom to produce and study double-mutants. Thus, he and his doctoral student, Simone Lombardi, verified that the competency gene acts together with the auxin and gibberellins hormones. A lower gibberellin content increases the plant’s competency – which, in the lab, will produce more stems from the leaf fragments. In addition to facilitating the production of seedlings for agricultural use, understanding these processes opens up the way to generate transgenic plants on a large scale.

The relationship between competency and development is very close and is manifested in several cycles of the plant’s life span. The cells of the leaves are normally not very competent: a leaf is like a dead-end alley; nothing will ever sprout from a leaf. The tips of the branches, however, contain cells that are responsible for the plant’s growth and for the on-going production of leaves, fruits, branches and flowers. In plants with increased competency, the cells of the leaves are more prone to generate other structures. This is why they develop more slowly and generate serrated or clipped edges or even edges subdivided into leaflets, in a crescendo that corresponds to the degree of competency. “A composite leaf is one step away from becoming a stem with open growth,” explains Peres, in contrast to the closed-end growth – that so-called dead-end alley – which characterizes leaves.

For everyone
The collection of mutant tomato plants allows researchers to investigate plant mysteries that had been previously inaccessible. But Peres has no intention of monopolizing the tomato plantation – he is at the disposal of other researchers and improvers – those who seek to improve the properties of plants with commercial value.

One of the genes found at Esalq induces the plant to produce meatier fruit – a parameter referred to as Brix level, which measures the content of soluble solids (organic acids and sugars) in the soft mass. The Brix level of a normal tomato plant is no higher than 5, but the team at Esalq was able to generate a mutant with a Brix level of 10, a historic result. In addition to being directly useful to improvers, the result shows the value of the tomato plant as a plant model. The same gene can affect the Brix level of other species with meaty fruit, such as oranges and coffee. The Arabidopsis plant, the model most frequently used in botanical studies, has dry fruit and this is why what is observed in this plant does not apply to species that produce fruit with commercial value.

Peres also uses his mutants as teaching material. In practical classes at university courses on plant physiology, students are guided on how to apply hormones in plants to see what kind of alterations are caused. “Hormones are very expensive,” the researcher explains, “so cultivating mutant plants for specific hormones is a lot cheaper.” In a course which is part of the graduate program in biology at Esalq, Peres gave his students mutant tomato seeds. Each group got a different, unidentified seed. Throughout the period of the course, the students planted the seeds and kept track of the growth of the plants. By combining the knowledge acquired in the theoretical classes with their own observations, the students had to find out, by the end of the semester, which function had been affected by the genetic alteration. “Things worked out very well, it was a stimulating exercise which involved the students,” says the professor enthusiastically.

The tomato plant has traveled a long, adventurous path from when it was discovered in the Andes until it became the Micro-Tom in the Piracicaba classrooms. The tomato plant was domesticated by the Aztecs, who lived from the XIV to the XVI century in what is now Mexico, where tomato plants did not grow spontaneously. When they colonized the Aztec Empire, the Spaniards took the plants with the red fruit back to Europe as ornamental plants, because they feared the tomato was toxic. The Italians did not resist the tomatoes’ appetizing look and incorporated them into their cuisine. They also developed improved varieties which then spread throughout the world. Nowadays, the tomato plant is a promising model that has opened up research routes that will probably help unveil the genetic and hormonal functioning of various species with economic value, and, who knows, of plants in general.

The Project
The genetic and biochemical bases of competency using the micro tomato plant as a model; Modality: Regular Line for Research Aid – Young Researcher; Coordinator: Lázaro Eustáquio Pereira Peres – Esalq/USP; Investment: R$ 224.126,65 (FAPESP)