By Emily Hubbell

A plant’s cell wall is the reason a plant looks the way it does. It creates form and acts as the first line of defense against harmful bacteria and fungi. Although the cell wall is a fundamental component of a plant, key aspects of this extracellular structure are still undiscovered.

Scientists know the plant cell wall is a matrix produced by of thousands of enzymes, each one helping to construct the wall’s web-like structure by synthesizing various sugars and proteins to form glycoproteins. But in most cases, researchers still don’t know which enzyme adds which sugar during this web-building process.

Ohio University researchers are working to identify the enzyme responsible for adding the first sugar in one of the plant cell wall’s glycoproteins, the Arabinogalactan-Protein (AGP). The National Science Foundation awarded the team $261,206 for the study.

Scientists have already converted material from the plant cell wall into biofuel through microbial fermentation, but they lack the research needed to optimize the process, said Allan Showalter, the grant’s principle investigator, professor of plant biology and NQPI member.

“The primary goal is to better understand how the plant cell wall is put together,” Showalter said. “But if we can understand how to modify this particular enzyme, it may influence the ability to remove lignin from the cell wall to get to the cellulose you need for biofuel production.”

The OU researchers—Drs. Showalter, Marcia Kieliszewski and Ahmed Faik—have determined that the first sugar in the plant cell wall, a galactose residue, binds to the AGP at the location where the amino acid Hydroxyproline (HYP) appears within its sequence. They are now trying to determine which enzyme or enzymes attach the galactose to the HYP.


The researchers grow the Arabidopsis plant in an OU greenhouse.


The team uses two techniques—proteomics and bioinformatics—to determine which enzyme(s) could cause this phenomenon. They have whittled down the list from thousands of enzymes to six strong candidates.

With proteomics, the researchers determine the amino acid sequences of numerous proteins contained in the cell membrane fractions associated with the transfer of galactose.

The next step is to identify corresponding genes and predict which genes will likely produce proteins that are targeted to the Arabidopsis plant’s Golgi apparatus with this enzyme activity.

One reason to use Arabidopsis is that all its genes have been sequenced, said Showalter. This means researchers know all the protein sequences, making the plant “essentially a library that can be searched.”


The Arabidopsis plant is a valuable tool for the researchers because its genes have been fully sequenced.

The other method, bioinformatics, lets the team compare plant wall enzymes to enzymes that perform similar roles in animals and other organisms. Specifically, they can search for protein domains associated with binding and transferring galactose residues.

Once they have selected candidate enzymes, the team tests each one in an assay. The researchers mix a HYP-containing AGP peptide with a radioactive version of galactose and then add the candidate enzyme to the reaction. After a two-hour incubation period, the scientists can determine whether the peptide is labeled with the galactose.