Study identifies genes that control quality of sugarcane

The objective now is to create a genetically modified plant that produces a type of lignin that is easier to separate from cellulose, easing the production process of second generation ethanol, explains Paulo Mazzafera (Unicamp), coordinator of the FAPESP Thematic Project.

By Karina Toledo

Agência FAPESP –  Although vital for the survival of plants, lignin, the structural material responsible for the rigidity, impermeability and resistance of vegetable tissue – is one of the major impediments to the use of sugarcane bagasse in ethanol production.

 

Given its strong bond to cellulose, this molecule impedes sugar found in the cellular wall from becoming hydrolyzed and released for fermentation. Although there is a pre-treatment capable of separating lignin from cellulose, it is expensive, laborious and can leave toxic residues for the fermentation microorganisms.

 

With the objective of facilitating the process and making production of cellulosic ethanol feasible, researchers from the Biology Institute at Universidade Estadual de Campinas (IB- Unicamp) have dedicated their time since 2009 to understanding how sugarcane synthesizes lignin. 

 

The proposal for the Thematic Project entitled “Control of lignin biosynthesis in sugar cane: many gaps still to be filled,” coordinated by Paulo Mazzafera, is to identify the genes involved in the metabolism of this polymer in order to develop a genetically modified variety that can synthesize a type of lignin that is more easily removed from bagasse.

 

The group has already identified four candidate genes for sugarcane transformation, which are directly linked to the quality of lignin produced by the vegetable. The researchers also found a fifth gene that if modified could alter the quantity of the substance in the plant. 

 

The main challenge now, according to Mazzafera is creating a genetically modified sugarcane strain that has the agronomical characteristics of a normal plant, with the height and width of the stalk. Read the researcher’s full interview with Agência FAPESP:

 

Agência FAPESP – What are the main objectives of the Thematic Project? 

Paulo Mazzafera – We began the study in 2009 with the objective of understanding how sugarcane synthesizes lignin and how this metabolism responds to certain types of stress, such as low temperatures or lack of water. This information will give us the tips to discover which genes could be modified to alter the plant’s lignin. 

 

Agência FAPESP – The idea was to create a genetically modified plant with less lignin? 

Mazzafera – We reached the conclusion that it is better to change the quality of the lignin than to reduce its levels in sugarcane.  Today, there are varieties that have between 4% and 10% lignin. If you reduce it too much, the plant won’t stand up. The varieties of sugarcane with lower lignin levels are used to make cellulose, but still pose some problems in second generation ethanol production. Our objective is to create genetically modified sugarcane that synthesizes a type of lignin that can be more easily removed from bagasse. Currently, the process requires a series of corrosive chemical products to separate the lignin from the cellulose and to release sugar from the cell walls to be fermented. Altering lignin could ease the process by generating less residue, diminishing the toxicity for yeast and increasing efficiency for production of second generation ethanol. 

 

Agência FAPESP – Have you managed to find candidate genes? 

Mazzafera – We identified four genes directly related to the quality of lignin produced by the plant. Lignin is a complex molecule formed basically by three compounds: coumaryl, coniferyl and sinapyl alcohol. When incorporated into lignin structure, they are called hydroxyphenyl, guaiacyl and syringyl units, respectively. Three of the identified genes direct biosynthesis of the syringyl unit. The next step now is to conduct genetic modification of sugarcane and alter the proportion of syringyl in lignin. The fourth gene found is a transcription factor that also regulates this process. We also found a fifth candidate gene responsible for production of an enzyme called laccase, but this is not directly linked to the quality of lignin, but instead the process of polymerization.

 

Agência FAPESP – And what are the consequences of meddling with this gene? 

Mazzafera – These three compounds are synthesized within the cell and are later thrown out and react forming a polymer known as lignin. But in order for this polymerization to occur, the compounds must be oxidated. And two enzymes can conduct this oxidation: peroxidase and laccase. Altering the production of these enzymes makes it possible to change the process of polymerization, which would affect the quantity of lignin synthesized. It is another possible path that we intend to study. Until now, the gene that produces laccase enzymes has been little studied.

 

Agência FAPESP – Will it be possible to develop genetically modified sugarcane under this Thematic Project? 

Mazzafera – That part was not originally envisaged in the project, but we can request renewal or submit a new proposal. The major challenge is ensuring that sugarcane maintain its agronomical characteristics. Although some people have already transformed sugarcane in Brazil, I have still not seen any case that shows that the plants produced are normal.

 

Agência FAPESP – What happens with modified plants?

Mazzafera – Normally they are shorter, sprout more and have finer stems. The problem is that sugarcane industry is equipped to harvest a certain type of plant. It must be erect and have stems that can accumulate large quantities of sugar. Having dwarf sugarcane and a giant harvester will not work. 

 

Agência FAPESP – Is it possible to know in advance whether the genes involved in lignin metabolism also influence other plant processes? 

Mazzafera – One of them uses a compound known as chlorogenic acid, which is abundant in all plants and altering this gene could change other metabolic steps. But we will only know for sure after making modified sugarcane.

 

Agência FAPESP – In addition to discovering candidate genes, are there other relevant results obtained under this Thematic Project?

Mazzafera – We published an article in Analytical Chemistry in which we describe a simple method to create a reference library that allows for comparisons and evaluating the type of lignin that is being produced by the plant. When those three compounds that form lignin are oxidated by peroxidase and laccase enzymes, they begin to polymerize spontaneously. This means that the monomers aggregate and form dimers, trimers, tetramers and pentamers, as so on – until it results in large molecule. The composition of this molecule could vary and this variation is related to the quality of lignin. This reference library can be useful in evaluating the results of research that aims to change the quality of lignin.

 

Agência FAPESP – Are there other published studies?

Mazzafera – There are two articles in Plant Physiology and Biochemistry on the role of peroxidase on the metabolism of sugarcane. Another study on the laccase enzyme was submitted to the Journal of Experimental Botany and is under evaluation. We are also preparing an article to describe all the genes involved in lignin metabolism in sugarcane. This is unprecedented. I believe that there are many studies to come.

 

The article Analysis of Soluble Lignin in Sugarcane by Ultrahigh Performance Liquid Chromatography–Tandem Mass Spectrometry with a Do-It-Yourself Oligomer Database (doi: 10.1021/ac301112y), can be read at pubs.acs.org/doi/abs/10.1021/ac301112y.

 

The article Suspension cell culture as a tool for the characterization of class III peroxidases in sugarcane (doi: 10.1016/j.plaphy.2012.10.015), can be read at www.ncbi.nlm.nih.gov/pubmed/23159486.

 

The article Enzymatic activity and proteomic profile of class III peroxidases during sugarcane stem development (doi: 10.1074/mcp.M112.019331), can be read at www.sciencedirect.com/science/article/pii/S098194281200071X.