Changes in Cell Aggregation in Arabidopsis thaliana Suspension Culture Following Knockout of GAUT Gene Family Members
Tatyana A Frankevich1, Natalya V Permyakova1, Yury V Sidorchuk1
1Federal Research Center Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, pr. Lavrentieva 10, Novosibirsk 630090, Russia.
Abstract:
Plant cells, particularly suspension cell cultures, represent a promising platform for the biosynthesis of biopharmaceutical proteins. However, one of the limitations of this system is the tendency of cells to form aggregates of varying sizes, which can restrict their productivity in terms of recombinant protein accumulation. The primary cause of such aggregation is likely related to intercellular adhesion, which is characteristic of plant cells grown in vitro. To assess the potential for reducing intercellular adhesion in in vitro plant cell cultures, we obtained two Arabidopsis thaliana cell lines carrying mutations in the GAUT7 and GAUT8 genes, which are involved in the biosynthesis of cell wall pectin. The GAUT7 mutant was generated by CRISPR/Cas9-mediated knockout of the target gene in a transgenic A. thaliana line carrying the gfp gene. The GAUT8 mutant cell line was derived from the A. thaliana Quasimodo1-1 mutant. We present a comparative analysis of these two in vitro cultured cell lines in terms of their aggregation behavior. The resulting mutant with a knockout in the GAUT7 gene had an altered cell culture phenotype. The GAUT7 suspension culture was characterized by a darker coloration, an increase in the number of large aggregates by 18%, and a decrease in the level of pectins, and the accumulation of recombinant GFP protein in the GAUT7 culture significantly decreased by 10.4%. The Qua1-1 culture showed the opposite results: a 20% decrease in the number of large aggregates, a high increase in biomass, and an increased level of pectins compared with the control and GAUT7. Thus, we have shown that a violation of pectin synthesis leads to different results depending on which GAUT family gene we knock out; intercellular adhesion decreased in the cell culture with a knockout of the GAUT8 gene. These data can be used to improve the properties of plant cellular expression systems of biopharmaceutically valuable proteins.
