Functional characterization of cell wall-related proteins of unknown function (CW-PUFs) in Arabidopsis thaliana

Abstract

Lignocellulosic biomass is an important feedstock for the bioeconomy, particularly for biorefinery and biomaterial application. This is due to the characteristic secondary cell walls composed of a matrix of cellulose and hemicellulose intricately linked to and rigidified by lignin. Studies have estimated 10-15% of ~27,000 protein-coding genes in the model herbaceous plant Arabidopsis thaliana are dedicated to cell wall development. However, conclusive experimental evidence validating cell wall functionality is only available for approximately 120 genes. This observation highlights a gap in our understanding which, poses a hindrance on biotechnology aimed at optimizing yield and reducing cell wall recalcitrance for efficient release of biopolymers. High-throughput omics technologies have generated inventories of cell wall-related genes, many of which are annotated as unknown (cell wall-related proteins of unknown function, CW-PUFs) due to absence of supporting evidence for biological and/or molecular function. The importance of CW-PUFs can be estimated by evolutionary conservation, co-expression with known genes (e.g. cellulose synthase genes) and experimental characterization. CW-PUFs may be classified into two groups, proteins containing conserved domains of unknown function (DUFs) and proteins of obscure features (POFs) lacking any recognized domains/motifs. The aim of this study was to identify candidate CW-PUFs in the fast-growing and economically important hardwood crop genus, Eucalyptus, and functionally characterize these genes in the model plant Arabidopsis. Using bioinformatics tools and approaches, xylem-expressed members of the DUF1218 gene family and a single POF gene were prioritized for functional characterization. To gain insight into its structure and suggested role in cell wall biology, a comparative genomics analysis of the DUF1218 gene family was performed. Approximately 284 non-redundant DUF1218-encoding genes were identified across 22 plant genomes revealing a land plant origin for the protein domain family. Furthermore, several DUF1218 family members putatively involved in cell wall biology were identified. We characterized the DUF1218 gene At4g27435 and showed that it is specifically expressed in interfascicular and xylem fibers, and loss-of-function results in a perturbation of lignin content in the cell walls of Arabidopsis. A second candidate, At1g31720, was also preferentially expressed in secondary cell wall depositing tissues. Due to potential functional redundancy, we included the most closely related family member At4g19370 and showed that both proteins are targeted to the cell periphery. No changes were seen in cell wall lignin content and growth in the loss-of-function and overexpression lines. However, we observed decreased rosette size, rosette fresh weight and stem length for the double homozygous mutant. Cell wall chemistry analysis revealed a decrease in the total lignin content and an increase in the syringyl/guaiacyl (S:G) monolignol ratio in the double mutant relative to the control plants. At1g31720 and At4g19370 were subsequently named MODIFYING WALL LIGNIN-1 (MWL-1) and MWL-2 respectively. Next, the role of an Arabidopsis POF gene (AtPOF1) in secondary cell wall biology was investigated. POF1 is Angiosperm-specific and a singleton in the sequenced plants. In agreement with characteristics of POFs, predicted DNA-, RNA- and protein-binding sites as well as regions of disorder were identified. AtPOF1 co-expressed genes were overrepresented in cell wall-related gene ontology terms including cell wall biogenesis. We showed that AtPOF1 expression is associated with xylem fibres, while the protein is nuclear-targeted. No changes in growth parameters were observed in loss-of-function or overexpression lines, however, secondary cell wall chemistry analysis revealed changes in the glucose content of the mutant line and total lignin content in the overexpression lines. This Ph.D. study identified and ascribed function to four new candidate CW-PUFs. We found a novel function for At4g27435 where previous attempts were inconclusive. Furthermore, we found two additional members of the DUF1218 family that function redundantly as contributors to secondary cell wall biology, specifically a lignin-related role. We provide insight into the role of a POF in secondary cell wall biosynthesis. The studies presented in this dissertation provides further understanding into cell wall biology and reveals new candidate genes for engineering plants with customised cell wall properties.