Starting to Gel: How Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls

For more than a decade, the Arabidopsis seed coat epidermis (SCE) has been used as a model system to study the synthesis, secretion and modification of cell wall polysaccharides, particularly pectin. Our detailed re-evaluation of available biochemical data highlights that Arabidopsis seed mucilage is more than just pectin. Typical secondary wall polymers such as xylans and heteromannans are also present in mucilage. Despite their low abundance, these components appear to play essential roles in controlling mucilage properties, and should be further investigated. We also provide a comprehensive community resource by re-assessing the mucilage phenotypes of almost 20 mutants using the same conditions. We conduct an in-depth functional evaluation of all the SCE genes described in the literature and propose a revised model for mucilage production. Further investigation of SCE cells will improve our understanding of plant cell walls

Monitoring Polysaccharide Dynamics in the Plant Cell Wall.

All plant cells are surrounded by complex walls that play a role in the growth and differentiation of tissues. Walls provide mechanical integrity and structure to each cell and represent an interface with neighboring cells and the environment (Somerville et al., 2004). Cell walls are composed primarily of multiple polysaccharides that can be grouped into three major classes: cellulose, pectins, and hemicelluloses. While cellulose fibrils are synthesized by the plant cells directly at the plasma membrane (PM), the matrix polysaccharides are produced in the Golgi apparatus by membrane-bound enzymes from multiple glycosyltransferase families (Oikawa et al., 2013). After secretion to the wall via exocytosis, the structures of the noncellulosic polysaccharides are modified by various apoplastic enzymes. In addition to polysaccharides, most plant cell walls contain small amounts of structural proteins such as extensins and arabinogalactan proteins.Cell walls are dynamic entities, rather than rigid and recalcitrant shells, that can be remodeled during plant development and in response to abiotic and biotic stresses. Cell expansion requires the deposition of additional material in the surrounding primary walls as well as the reorganization and loosening of existing polymers to allow for wall relaxation and controlled expansion (Cosgrove, 2005). The latest model of the primary wall structure proposes that cellulose-cellulose junctions only occur at a limited number of biomechanical hotspots, where protein catalysts must act selectively to initiate wall loosening (Cosgrove, 2018). In tissues undergoing growth, the recycling of polysaccharides via a suite of enzymes can contribute to the construction of elongating walls (Barnes and Anderson, 2018). Once elongation ceases, some cells deposit thick secondary walls that incorporate additional polysaccharides. Many secondary walls are impregnated with the polyphenol lignin and thereby become relatively fixed structures that exclude water and resist hydrolysis

TRM4 is essential for cellulose deposition in Arabidopsis seed mucilage by maintaining cortical microtubule organization and interacting with CESA3552

The differentiation of the seed coat epidermal (SCE) cells in Arabidopsis thaliana leads to the production of a large amount of pectin‐rich mucilage and a thick cellulosic secondary cell wall. The mechanisms by which cortical microtubules are involved in the formation of these pectinaceous and cellulosic cell walls are still largely unknown. Using a reverse genetic approach, we found that TONNEAU1 (TON1) recruiting motif 4 (TRM4) is implicated in cortical microtubule organization in SCE cells, and functions as a novel player in the establishment of mucilage structure. TRM4 is preferentially accumulated in the SCE cells at the stage of mucilage biosynthesis. The loss of TRM4 results in compact seed mucilage capsules, aberrant mucilage cellulosic structure, short cellulosic rays and disorganized cellulose microfibrils in mucilage. The defects could be rescued by transgene complementation of trm4 alleles. Probably, this is a consequence of a disrupted organization of cortical microtubules, observed using fluorescently tagged tubulin proteins in trm4 SCE cells. Furthermore, TRM4 proteins co‐aligned with microtubules and interacted directly with CELLULOSE SYNTHASE 3 in two independent assays. Together, the results indicate that TRM4 is essential for microtubule array organization and therefore correct cellulose orientation in the SCE cells, as well as the establishment of the subsequent mucilage architecture

Modular biosynthesis of plant hemicellulose and its impact on yeast cells

Abstract Background The carbohydrate polymers that encapsulate plants cells have benefited humans for centuries and have valuable biotechnological uses. In the past 5 years, exciting possibilities have emerged in the engineering of polysaccharide-based biomaterials. Despite impressive advances on bacterial cellulose-based hydrogels, comparatively little is known about how plant hemicelluloses can be reconstituted and modulated in cells suitable for biotechnological purposes. Results Here, we assembled cellulose synthase-like A (CSLA) enzymes using an optimized Pichia pastoris platform to produce tunable heteromannan (HM) polysaccharides in yeast. By swapping the domains of plant mannan and glucomannan synthases, we engineered chimeric CSLA proteins that made β-1,4-linked mannan in quantities surpassing those of the native enzymes while minimizing the burden on yeast growth. Prolonged expression of a glucomannan synthase from Amorphophallus konjac was toxic to yeast cells: reducing biomass accumulation and ultimately leading to compromised cell viability. However, an engineered glucomannan synthase as well as CSLA pure mannan synthases and a CSLC glucan synthase did not inhibit growth. Interestingly, Pichia cell size could be increased or decreased depending on the composition of the CSLA protein sequence. HM yield and glucose incorporation could be further increased by co-expressing chimeric CSLA proteins with a MANNAN-SYNTHESIS-RELATED (MSR) co-factor from Arabidopsis thaliana. Conclusion The results provide novel routes for the engineering of polysaccharide-based biomaterials that are needed for a sustainable bioeconomy. The characterization of chimeric cellulose synthase-like enzymes in yeast offers an exciting avenue to produce plant polysaccharides in a tunable manner. Furthermore, cells modified with non-toxic plant polysaccharides such as β-mannan offer a modular chassis to produce and encapsulate sensitive cargo such as therapeutic proteins. Graphic abstrac