Characterization of a Glycan Exo-Hydrolase that Shows a Biphasic Expression in the Course of an In Vitro Culture of Photoautotrophic Oxybasis rubra Cells

Abstract

Like plant leaves, suspension cultures of photoautotrophic Oxybasis rubra Fuentes-Bazan (syn. Chenopodium rubrum L.) cells pass through distinct developmental phases when grown under CO2 as the sole carbon source: an initial cell division phase of 4 weeks, a stationary phase of another 4 weeks and an aging phase (3-4 weeks) when the cell senesce and finally die. These phases are reflected by differential gene expression. A gene that was strongly expressed in the course of the stationary phase but much lesser during the exponential growth phase of the cell culture was isolated from a cDNA-library of stationary cells and completed by 5’-RACE. From homology analysis, the gene was tentatively identified as glycan exo-hydrolase (Oxybasis rubra glycan exo-hydrolase, OrGEH). Heterologous expression in E. coli yielded a protein with a preference to hydrolyze the s-D-galactopyranoside, s-D-fucopyranoside and s-D-glucopyranoside of the corresponding artificial p-nitrophenyl substrates. Possible function of the protein in cell-wall metabolism was confirmed by extracellular localization of the GFP-fusion protein in transformed Nicotiana benthamiana leaves, by an N-terminal signal sequence for the secretory pathway and by the positive response of gene expression to auxin. Suspension cultured OrGEH RNAi-lines showed not only strongly reduced cell division activity but also lack of the stationary phase during which the cells usually double their biomass by extension growth. Although the natural substrate of the protein is not known we propose a function of OrGEH in the processing of cell wall components during cell division and cell extension growth.