Heterologous Expression and Functional Analysis of Plantago GT61 and DUF579 Genes in Arabidopsis thaliana

Abstract

Mucilage released from Plantago ovata seed (psyllium) has been used for maintaining human health as a dietary fibre supplement. Heteroxylan is the main component, and its substitution affects solubility and viscosity of the end product. However, little is known about genes involved in xylan substitution so phylogenetic and transcript information were used to identify candidate genes in the GT61 and DUF579 families and their functions were tested in the model plant Arabidopsis thaliana. Plantago GT61_7, driven by a seed-coat promoter (ProDP1) was transformed into Arabidopsis using a floral dip and spray method. Ruthenium red staining of wild-type and T2 seeds from multiple independent transgenic lines showed a significant difference in the thickness of the adherent mucilage layer. The difference in mucilage phenotype suggests that GT61_7 may have a role in xylan substitution that affects seed coat adherence. This preliminary result needs to be examined using immunolabeling and monosaccharide analysis. For the DUF579 gene AT1G71690, a genome editing approach was adopted. Three single guide RNAs were designed using online tools and in silico analysis was performed to predict any changes in coding and protein sequences by each guide RNA. To test them in vitro, the CRISPR/Cas9 constructs were successfully delivered to protoplast cells using the Transient Expression in Arabidopsis Mesophyll Protoplast (TEAMP) method. However, an analysis using Tracking of Indels by Decomposition(TIDE) showed no evidence of edits in the DUF569 genomic DNA extracted from the protoplasts. Increasing the transfection efficiency or redesigning the sgRNA could lead to improved CRISPR/Cas9 activity.