In times of stress

Under extreme conditions, a plant used in tequila production feeds on bacteria that live within it

Blue agave, which typically grows in regions with poor, sandy soil, is the raw material for tequila

Léo RamosBlue agave, which typically grows in regions with poor, sandy soil, is the raw material for tequilaLéo Ramos

Tequila, the famed Mexican distilled beverage, is made from the pulp of the blue agave (Agave tequilana), a plant whose leaves are long, hard and spiny. It is a desert plant that grows in poor, sandy soils, owing in part to nutrients produced by bacteria that live harmoniously within its cells. But in times of extreme need, such as long periods of drought or intense sun, the blue agave sacrifices these bacteria and feeds on them in order to survive. “The plant literally consumes its own bacteria to stay alive,” explains Paolo Di Mascio, a biochemist with the Chemistry Institute at the University of Sao Paulo (IQ-USP).

Di Mascio was a member of an international team that conducted a number of experiments at IQ-USP and at Rutgers University in the United States, demonstrating that the blue agave digests the Bacillus tequilensis bacteria normally found in its cells. That strategy enables the plant to absorb at least some of the nutrients it needs to get through periods of deprivation. The findings from that study, which was published in the journal Scientific Reports in November 2014, also reveal that, even when nutrients are abundant, the blue agave benefits from the partnership with B. tequilensis: the presence of the bacterium makes the agave grow faster, as much as tripling its production of biomass.

Other recent research studies have indicated that certain species of bacteria collaborate to achieve plant growth and control agricultural pests. “We are looking for microorganisms that can substitute for or reduce the use of fertilizers and pesticides,” says Miguel Beltrán-García, a researcher at the Autonomous University of Guadalajara, Mexico, who headed the agave studies.

In the lab: leaves are macerated...

Léo RamosIn the lab: leaves are macerated…Léo Ramos

Beltrán-García conducted the experiments in 2013 during a year-long stay at IQ-USP at the invitation of Di Mascio, with whom he has done collaborative research for more than a decade. Working with another international team, they identified a biochemical pathway by which the Mycosphaerella fijiensis fungus damages banana plants. The experiments, described in the journal PLOS One in 2014, indicated that when sunlight hits the pigment melanin produced by the fungus, it triggers photochemical reactions that produce molecules of excited oxygen and lead to cell death in the plant leaves, which turns them dark. The researchers are continuing to study the pest, known as black Sigatoka, in order to better understand how a liquid solution developed by Beltrán-García and his colleagues was able to control the disease in commercial plantations in Mexico. The solution is made of a mixture of bacteria obtained from the banana plants themselves, and in addition to its use as a pesticide, it also serves as a fertilizer.

As a result of his research on the banana pest, Beltrán-García is concerned about the future of the blue agave, one of the principal agricultural products of the Mexican state of Jalisco, where the city of Guadalajara and the town of Tequila are located. Tequila is not the only product derived from blue agave that is manufactured on an industrial scale. The plant also produces a syrup that, after cooking, is sweeter than honey, as well as inulin, a sugar used in food production. “Blue agave production is suffering the effects of fungal and bacterial diseases and of insect attacks,” Beltrán-García explains.

Hungry for nitrogen
In an attempt to increase productivity, farmers use excessive amounts of fertilizer–a worldwide phenomenon in agriculture–which also leads to undesirable consequences. Fertilizers contain nitrate salts and ammonium chloride, a source of nitrogen, which is one of the most essential plant nutrients. That chemical element is a component of protein, DNA and the molecule chlorophyll, the pigment responsible for the photosynthetic reaction that feeds plants. The problem is that only half the nitrogen in fertilizers is absorbed by plants. The remainder goes into the environment, where it can harm soil quality and pollute distant ecosystems when it is carried far way by wind or water.

...and cut up...


Because of these effects, scientists are looking for alternatives to fertilizers. One such alternative are bacteria that help plants extract nitrogen. They convert nitrogen from air (inert for most organisms) and from other compounds into molecules that plants can absorb, such as ammonia. Other bacteria break down dead organisms and make nitrogen-based compounds available to plants.

More recently, scientists have noted that plants also acquire nitrogen with the help of another type of microorganism, endophytic bacteria, such as Bacillus tequilensis, which live within plant cells in harmony with their hosts. No one knows, however, how the nutrients produced by these bacteria–whether in the soil, in roots or in plant cells–are utilized by the agave.

“It’s difficult to study the flow of nutrients from microbes to plants,” says James White, an expert in plant-microorganism interaction based at Rutgers University, who did collaborative research with Beltrán-García and Di Mascio on the black Sigatoka study. “That is probably why no one has done this type of experiment,” says White, who also took part in the blue agave research with his colleague Monica Torres.

White, who studies grasses that play host to endophytic bacteria, believes that plants can digest microorganisms by producing oxygenated water (H2O2). The chemical formula of H2O2 is similar to that of water: it has an extra oxygen atom, which tends to react with other molecules. White conjectures that the oxygenated water released by the plant destroys the endophytic bacteria and breaks down their large molecules into smaller molecules, which can then be utilized by the plant cells. “We have evidence that this occurs in some species, but we believe this process can occur throughout the plant kingdom,” he says. “The unanswered question is knowing how important nitrogen from endophytes is for the plant.” The answer probably varies from one plant species to another, and according to the prevailing circumstances.

...for two types of analysis that gauge nitrogen-15 acquisition

LÉO RAMOS…for two types of analysis that gauge nitrogen-15 acquisitionLÉO RAMOS

In one experiment, White and Torres used a microscope to observe cells of blue agave root cast oxygenated water onto B. tequilensis bacteria inoculated into the plant. The test, however, left one area of uncertainty: Is the release of oxygenated water the plant’s defense mechanism against excess bacteria, or is it a way of acquiring nutrients?

Following the isotope
Di Mascio then had the idea of growing B. tequilensis in the laboratory, and feeding the bacteria a special nitrogen that could be tracked and later detected in molecules produced by the plants. He and his team gave the bacteria a type of nitrogen that has a weight of 15 atomic mass units, unlike most nitrogen atoms found in nature, which have an atomic mass of 14.

At IQ-USP, Fernanda Prado, Kátia Prieto and Marisa Medeiros of the Biochemistry Department, and Lydia Yamaguchi and Massuo Kato of the Basic Chemistry Department, fed the bacteria containing nitrogen-15 to blue agave seedlings grown under controlled conditions. In one experiment, the seedlings were removed from the greenhouse to be washed and sterilized once a week. They then spent several hours in a bottle containing only sterile sand and a solution of B. tequilensis, which simulated a nitrogen-poor environment. The researchers increased the environmental stress by leaving the agave under a very intense light.

054-057_Tequila_230Six months later, the researchers collected the leaves that had sprouted during that time and analyzed them in mass spectrometers, instruments that can distinguish the two types of nitrogen. They found nitrogen-15 in amino acids (the building blocks of protein) as well as in DNA and pheophytin, a molecule derived from chlorophyll. “Pheophytin is typically found in the plant and does not exist in the bacterium,” Di Mascio explains. “Finding pheophytin with nitrogen-15 is proof that atoms of the bacteria absorbed by the roots ended up in a molecule created by the plant.”

In another experiment, the researchers compared growth in seedlings that did not receive weekly doses of B. tequilensis with those that received live or dead bacteria. The seedlings fed with live bacteria grew twice as much as those that received dead bacteria, and three times as much as those fed with a mineral solution containing nitrogen. The finding suggests that, in addition to supplying nitrogen, the B. tequilensis that live in blue agave plants produce plant growth hormones called auxins.

Brazil-based studies conducted on sugarcane have shown that inoculation with endophytic bacteria can considerably accelerate plant growth. In partnership with Antonio Figueira and Layanne Souza of the Luiz de Queiroz College of Agriculture at USP, Di Mascio and Kátia Prieto plan to conduct experiments on sugarcane that are similar to those done on blue agave. “Working with sugarcane is harder, because in the laboratory the seedlings take a lot of added sugar to grow,” Di Mascio explains. “The sugar raises the risk of contamination by other bacteria and fungi, which can ruin the experiment.”

Chanyarat Paungfoo-Lonhienne, a researcher at the University of Queensland, Australia, says that the agave finding “invites us to investigate whether that is also a means of nutrition for other plants.” In 2010 she led the first study to show that plants–in this case, tomato and Arabidopsis thaliana, a model plant used in research–are capable of digesting invasive bacteria and fungi. In Paungfoo-Lonhienne’s view, understanding how these bacteria interact with plants and associating this combination with other organic techniques could lead to interesting findings. “It could reduce the use of fertilizers, if not replace them completely.”

1. Singlet molecular oxygen and peroxides in chemical biology (2012/12663-1); Grant mechanism Thematic project; Principal investigator Paolo Di Mascio (IQ-USP); Investment R$3,408,783.02 (FAPESP).
2. Redoxome – Redox Processes in Biomedicine (2013/07937-8); Grant mechanism Research, Innovation and Dissemination Centers (RIDC); Principal investigator Ohara Augusto; Investment R$22,604,697.96 (FAPESP – entire project).

Scientific article
BELTRÁN-GARCÍA, M. J. et al. Nitrogen acquisition in Agave tequilana from degradation of endophytic bacteria. Scientific Reports. V. 4, No. 6.938. Nov. 6, 2014.