Researchers uncover defense code for sugarcane

Mechanisms used by sugarcane plants to resist invasion by microorganisms and enzymatic hydrolysis are identified. Discovery paves the way for production of second-generation ethanol.

By Elton Alisson, in Beijing

Agência FAPESP – One of the principal bottlenecks for the production of second-generation ethanol (obtained from biomass) is the extraction of energy from the chemical bonds in the polysaccharides of the cell walls of plants such as sugarcane.

According to experts on the subject, the reason for this is that the plants’ cell walls have a highly complex organization, with a number of ramifications. This kind of organization makes them resistant to enzymes that can carry out the process of breaking the chemical bonds of the polysaccharides (hydrolysis) and enable sugar extraction.

A group of researchers at the National Institute of Science and Technology of Bioethanol – one of the national institutes of science and technology (INCTs) established in the state of São Paulo and supported by FAPESP in partnership with the National Council for Scientific and Technological Development (CNPq) – has uncovered some of the mechanisms that make the cell walls of sugarcane resistant to enzymatic hydrolysis.

The findings of their research, which will appear in a paper accepted for publication in the journal Bioenergy Research, were presented on April 17 during FAPESP Week Beijing – Brazil-China Scientific Collaboration in Beijing, China.

The event, jointly promoted by FAPESP and Peking University, brought together researchers from the two countries on April 16-18 to discuss research in the fields of materials science, the environment, renewable energy, agriculture, life sciences, medicine and health, in the interest of developing scientific collaboration.

“We are now able to understand in part what we call plant cell wall architecture, that is, how polymers aggregate and form a complex structure that does not happen by chance,” said Marcos Buckeridge, a professor at the Biosciences Institute of the University of São Paulo and coordinator of INCT/Bioethanol, in comments to Agência FAPESP.

“This led us to the hypothesis that the plant cell wall has a glycome code that causes some parts of it to be open to enzymatic hydrolysis, while other parts are not,” he explained.

The architecture of plant cell walls involves chains of cellulose microfibers that interact and form a group of 36 molecules called microfibrils, which aggregate to form macrofibrils.

The macrofibrils form a barrier that prevents water from entering the plant cell wall, and make it extremely difficult to break the chemical bonds of the polysaccharides in the cell walls.

In the case of sugarcane, the researchers discovered that the plant cell wall is composed of a group of seven microfibrils cross-linked by hemicelluloses.

This formation makes enzymatic hydrolysis of the plant cell wall even more difficult because it reduces the possible area of enzyme action, the researcher explained.

“This poses a great challenge for cellulose hydrolysis, because it can only be broken on the surface,” Buckeridge said.

Programmed death

The INCT/Bioethanol researchers discovered, however, that the root of sugarcane undergoes a process similar to that observed in plants such as the papaya tree.

As a papaya fruit ripens, the structure of its cell walls changes, softening it and making it easier to break open in order to facilitate seed dispersal.

In the case of sugarcane, the researchers found that during ripening, the plant’s cell walls undergo modifications in which spaces are formed for air circulation to improve performance.

“This type of mechanism, known as aerenchyma, is frequently used by plants growing in flood areas. And even though sugarcane is not subject to constant flooding, it too displays this phenomenon,” Buckeridge noted.

According to Buckeridge, aerenchyma in sugarcane root is initiated by a hormone signal associated with the balance between the hormones ethylene and auxin.

Upon receiving this hormone signal, part of the plant root initiates a programmed cell death in which the cell’s mitochondria begin to collapse, triggering sequential processes of cell separation and expansion, hemicellulose hydrolysis, and finally, cellulose hydrolysis.

In each of these stages, there is a set of enzymes used by the sugarcane to alter its own cell walls. These include expansin, a protein known for its ability to break the hydrogen bridge bonds and, as a result, separate the hemicellulose from the cellulose; endopolygalacturonase, which carries out cell separation; and spectrin, which degrades the polymers that hold the cells together, Buckeridge explained.

Through the use of sophisticated methods of cell wall analysis, the researchers characterized the aerenchyma phenomenon in sugarcane and identified the genes and enzymes that initiate the process.

The idea now is to carry out the sugarcane transformation with the identified genes to evaluate the effects of modifying the plant with some of these proteins, said Buckeridge. “We are assessing whether we’ll be able to do that now on the whole plant,” he said.

One gene that is a candidate for use in sugarcane transformation to increase the efficiency of enzymatic hydrolysis is RAV – a gene known as a transcription factor that initiates senescence in plant tissues.

The researchers are currently evaluating whether this gene is linked in the sugarcane genome to the endopolygalacturonase enzyme and whether it initiates the cell separation process.

“Our goal is to do the sequencing of a group of sugarcane genes that will enable us to develop a strategic plan for “engineering” the plant cell wall so that it has more parts open, where the enzymes can act and break the polysaccharide bonds, and fewer regions that interact with one another and have ramifications that prevent enzymatic hydrolysis,” Buckeridge explained.

“We plan to leave the sugarcane well prepared, with the cell walls ‘softened,’ to lower the cost of the cocktail of enzymes and microorganisms used in plant hydrolysis, or even eliminate that stage of pretreatment,” he asserted.

Evolutionary strategy

According to Buckeridge, the glycome code was a strategy developed by plants as they evolved, to prevent invasion by pathogenic (disease-causing) microorganisms and keep the plant system stable.

“If the glycome code were easily broken, an emerging microorganism, for example, could invade any cell wall and hydrolyze it. And as a result we would risk extinction of all the plants,” he reckoned.

In addition to its applications in bioenergy, the mechanism can be useful for other areas of agronomic research, such as pest control or improving fruit, for example, the researcher said.

“Through the glycome code, researchers in the field of agriculture, for example, can control the texture and ripening of plant fruits, for example,” he noted.