Michelle Laterrade
Faculty Advisor: Jocelyn Rose
Year: 2015

Characterization of the Arabidopsis thaliana Glycosyl Hydrolase family 10(GH10)

Project Summary

Carbohydrate-active enzymes serve as catalysts in the metabolism and modification of glycosidic bonds. In plants, glycosyl hydrolases (GHs), a main class of carbohydrate-active enzymes, catalyze the biosynthetic and hydrolytic reactions required for cell-wall synthesis, modification, and disassembly.  This study aimed to characterize members of the Arabidopsis thaliana GH family 10 (GH10), which are annotated as endo-β-1, 4 xylanases (EC 3.2.1.8). From this, we infer that they act on xylan, a major hemicellulosic polysaccharide in primary and secondary plant walls. In addition to their amino-acid sequence similarities, the GH10 enzymes have a varying number of phylogenetically related carbohydrate-binding modules (CBMs) preceding their catalytic domain. Interestingly, some of the GH10s lack a predicted secretory signal peptides, which suggest a non-classical secretion pathway if they are indeed localized in the cell wall. This project employed a variety of molecular, genomic, and bioinformatic approaches to characterize the A. thaliana GH10 family members, AtXyn1, AtXyn2, AtXyn3, and AtXyn4. Xylanase proteins fused to a red fluorescent reporter protein (tdTomato) and various organelle markers fused to a green fluorescent protein were transiently expressed in onion cells to confirm their sub-cellular localization. Additionally, the organ and tissue localization of these enzymes was evaluated using a β-glucuronidase reporter assay. Lastly, A. thaliana T-DNA insertional mutants of AtXyn1, AtXyn2, AtXyn3, and AtXyn4 were analyzed for developmental and biomechanical phenotypes under various growth conditions.

My Experience

I feel tremendously blessed to have an opportunity to work with the staff and faculty at The Boyce Thompson Institute.  The program staff created a family-like atmosphere from day one, and the scientists continuously challenged and intrigued us with scientific presentations and discussion. As a young scientist, it is important to be surrounded by a team of people that challenge you to think critically and independently, which BTI provides devoid of pressure and intimidation.

In addition, working with the Rose lab allowed me to acquire a great deal of experience in molecular and genetic research from a different approach than I had studied before. Rigorous hours of experiments proved worthwhile, as I became captivated with results and possibilities of scientific discovery. I was delighted to be surrounded by enthusiastic scientists who continually enriched my understanding of everything from my current research outlooks to how to deal with daunting decisions.