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Open, Sesame!

Bacteria contend with plants to control the pores of the leaves

Nature magazineSurface of a leaf seen in the microscope: stomata fulfill vital functions for plantsNature magazine

When they reach the plants, the Pseudomonas syringae bacteria straight away look for open doors by which they can enter and cause lesions in the leaves and in the branches. The doors are the stomata, microscopic pores that have the capacity to open or to close. But the plant detects the enemy and rapidly blocks its entrances. With the doors closed, the bacteria have no way of entering, but they do not give up. They discover the key to open them and attack their victim.

It seems like a science fiction battle, but it is real. Closing the stomata is an innate response of the plants that restricts invasion by bacteria. The immunological function used to be unknown in these structures that are responsible for the plants’ gaseous exchange and transpiration. The discovery was made by Brazilian biologist Maeli Melotto, currently under contract as an assistant researcher at Michigan State University (MSU), United States.  “The stomata represent the first barrier against bacterial infection, and a substance released by the bacteria, coronatine, blocks this defense”, sums up Maeli, the first author of the article published in the September issue of the scientific magazine with these results.

The finding is an important step in the knowledge about stomata. The defense mechanism on the surface of the leaves was even unknown to a large extent because of the inoculation method, the most common one in laboratories. To study the plant immune system, researchers inject the infectious agent (viruses or bacteria) directly inside the leaf.  Maeli’s quest was driven by the observations of other researchers made ten years ago, which showed the inefficiency of some bacteria in causing infection when inoculated into surface of the leaves, instead of inside them. “As in laboratory conditions the bacteria only enter the leaves by the stomata, I began to study whether it was these that protected the leaves against invasion”, she says.

The team from the laboratory coordinated by Sheng Yang He used the Arabidopsis plant, a sort of version of the laboratory mouse, to study the molecular and blocking mechanisms that allow the plants to avoid bacterial infections. The group exposed Arabidopsis leaves to a lineage of P. syringae that is harmful to plants. The plants had between 70% and 80% of their stomata open, a situation typical of light conditions favorable to photosynthesis. But the bacteria did not remain at a loose end: they distinguished the closed stomata from the open ones and agglomerated around them like passengers that push each other to get into the subway train before the bell goes, in the rush hour.

The plant does not warn with a bell, but on detecting the invaders, the leaves in the experiment reacted by closing the stomata: in two hours, only 30% of them remained open. The defense reaction was transitory. Three hours afterwards, despite the bacteria remaining there, there were once again just as many stomata open as before the attack. What leads them to open up is a substance released by the bacteria called coronatine, which has on the leaves the effect of “Open, Sesame”, the magic words that Ali Baba used to enter the cave of the 40 thieves.

The researchers also show that the reaction to bacteria is a generalized reflex that is independent of the damage caused by the invader. The stomata also close when exposed to Escherichia coli, a bacterium that attacks human beings but is inoffensive to plants. However, unlike what they discovered with P. syringae, Maeli and her colleagues observed that E. coli does not have a mechanism that induces the reopening of the doors. As it is not a specialist in attacking plants, this bacterium has not discovered the password that ensures entry.

Discrete protagonists
Despite being essential for the life of the plants, little is yet known about how stomata work. The schoolbooks say that they open during the day to absorb carbon dioxide, which will be transformed into energy by the process of photosynthesis. But the problem faced by the plants is that they lose water through the open pores, a phenomenon known as transpiration. For this reason, the danger of drying out obliges the plant to close them in the hottest hours. The same goes for when it is not possible to do photosynthesis, which requires sunlight, but new data is putting this view in doubt. “There was a dogma that the plants close the stomata during the night, but we are now seeing that the story is different”, says Rafael Oliveira, a botanist from the Agriculture Nuclear Energy Center (CENA) of the University of São Paulo (USP) in Piracicaba.

Recent studies suggest that the stomata may also be an alternative to roots in the absorption of water. Stephen Burgess and Todd Dawson, from the University of California at Berkeley, United States, showed in 2004 that the Californian sequoias drink water through the leaves. It is still not proven that the stomata are the maelstroms in this case, but Rafael Oliveira, from USP, believes that this is the most plausible hypothesis. The other mechanisms considered, could be, according to him, slower than he and his colleagues observed.

Oliveira did his doctoral studies at Dawson’s laboratory and is investigating whether in Brazil the same thing that was discovered with the sequoias. His studies have shown that the plants from the Cerrado and from the Amazon also absorb water through the leaves. The botanist believes that the mechanism is a generalized response of plants to the availability of water in the environment. The case of the sequoias is exemplary, since they are trees of a large size that live in a region with month after month of drought, with thick daily cloud during this period. There is accordingly more water in the air than in the ground. To ascertain how the water enters the leaves, experiments will have to be done in the laboratory, such as using substances that induce the closing of the stomata and to compare the foliar water absorption with that of plants that have not been manipulated.

Plant mystery
It is not yet known how the plants detect the environmental conditions, nor how they solve the contradictory indications that the sun, the humidity of the air, the immune system and the bacteria provide to close or to open the stomata. But the defense reaction appears to have great weight in the decision: Maeli carried out her experiments in bright light, when the plants do photosynthesis and for this reason have their stomata open. Even so, about 80% of the stomata open closed up in the presence of the bacteria. “This fact suggests that the plant reduces photosynthesis when attacked by pathogens”, she concludes. In an earlier work with beans, published in the Genome magazine in 2005, she observed that genes connected with photosynthesis reduce their activity when the plants are attacked by some infectious agent. These results indicate that the closing of the stomata is not an isolated phenomenon, but part of a system that coordinates the plant’s vital functions with its defense.

The discovery having been made in Arabidopsis, it has to be demonstrated that the same is valid for other systems. The use of biological models, like Arabidopsis for plants, laboratory mice for mammals or fruit flies for insects, makes it possible for research to advance more rapidly. After discovering a mechanism in one of the species, it is possible to direct the studies in search of specific properties of other organisms. Maeli also examined tomato plants and tobacco plants, which showed the same kind of response.

For the time being, the group from the University of Michigan is going to invest in understanding better the mechanism that it discovered. “We intend to study in greater detail the molecular components involved in the stomatal defense and how the coronatine acts to disarm it. We also want to understand whether this defense is effective against other pathogenic bacteria that enter into the leaf, chiefly by the stomata”, Maeli says.

In a tropical forest, the leaves house an immense diversity of microorganisms (up to 600 species on a single leaf) and complex processes of interaction (please see this year’s July issue of Pesquisa FAPESP). The world of the leaves goes so far as to have a name amongst the specialists, the phylosphere. But there is still much to be tamed in this universe.