Daniel J. Cosgrove

Plant cell wall expansion is essential for plant growth, with expansins promoting wall creep during diffuse growth. Expansin’s role in tip growth, such as root hair elongation, is less clear. Here, we used CRISPR/Cas9 to knock out root hair–specific α-expansins EXPA7 and EXPA18 in Arabidopsis thaliana , which abolished root hair elongation. Complementation with expansin genes from various clades (driven by the EXPA7 promoter) revealed functional differences: Some fully restored root hair growth, others only partially restored growth, while others failed, notably EXPA13 (clade-VIII) and EXPA20 (clade-IX)—both lacking a conserved Asp considered essential, but not sufficient, for expansin-induced wall enlargement. Phylogenetic analysis suggests loss of this Asp may have predated angiosperms. Mutation of this Asp in EXPA7 confirmed its necessity for wall loosening, but its restoration in EXPA13 did not restore activity. Other expansin families (EXPB, EXLA, and EXLB) failed to restore root hair growth. Chimeric fusions of EXPAs with mCherry revealed differences in trafficking patterns and wall binding among EXPA clades. These results demonstrate functional differences among EXPA clades and among expansin families, establishing a genetic platform to analyze functionalities (trafficking, binding, activity) of EXPA and other wall-modifying proteins during root-hair tip growth.

The hierarchical organization of cellulose microfibrils and their intimate interactions with hemicelluloses such as xylan underpin the exceptional mechanical performance of plant cell walls. However, translating these biological design principles into sustainable nanocellulosic materials remains limited by conventional cellulose nanofibril production routes, which rely on harsh chemical treatments that disrupt the native cellulose–hemicellulose architecture. Here, we present an optimized isolation strategy for holocellulose nanofibrils (hCNFs) that preserves native cellulose structure, xylan substitution and conformation, and cellulose–xylan interactions. Using wild-type Arabidopsis thaliana , a xylan glucuronidation-deficient gux1/2 mutant, and Brassica napus straw as model systems, we systematically elucidate how xylan content and substitution pattern govern nanofibril isolation, interfacial interactions, and macroscopic properties. Two-dimensional ¹³ C magic-angle spinning NMR demonstrates retention of native cellulose glucosyl environments, the presence of two-fold and three-fold helical xylan conformations, and cellulose-associated two-fold helical xylan. Cryogenic transmission electron microscopy reveals fibril widths of ∼3 nm, consistent with elementary cellulose Iβ microfibrils. We show that xylan glucuronidation regulates colloidal stability, hydration behavior, and interfibrillar cohesion, whereas xylan content controls nanofibrillation efficiency. These multiscale structural features translate directly into moisture sorption, thermal behavior, and mechanical performance. Notably, Brassica napus hCNF films exhibit exceptional strength and extensibility, surpassing many chemically modified CNF systems. This work demonstrates that preserving the native cellulose–hemicellulose architecture enables high-performance, sustainable nanocellulosic materials without chemical reconstruction.

Plant primary cell walls are dynamic supramolecular assemblies composed of layered cellulose, hemicellulose, and pectin, progressively built through synthesis and secretion. However, the specific architectural features and structural components sufficient to endow the mechanical properties of the wall remain unclear. Here, we construct a minimal synthetic spherical shell and compare its structural and mechanical properties to those of a plant single-cell system. To eliminate complexities from intercellular connectivity and developmental history, we exploit the ability of plant protoplasts to regenerate cell walls de novo. Compression tests of regenerating protoplasts between parallel plates reveal that wall stiffness increases with wall thickening over time. Despite differences in assembly pathways, architecture, and composition, the synthetic shell exhibits a similar thickness-dependent modulus and similar material stiffness. The synthetic shell, mainly composed of pectin and cellulose nanofibers, mirrors the mechanical behavior of regenerating primary cell walls, suggesting that these components play a major role in conferring key mechanical properties in the limit of compressive small deformations. Extending this comparative approach should allow similarities and differences in component interactions in controlling wall behavior to be identified.

Plant cell wall enlargement is fundamental to crop productivity and its sensitivity to drought ¹ . Tip growth and diffuse growth are contrasting wall enlargement patterns often proposed to be limited by different processes: localized secretion and remodeling of pectins for tip growth versus loosening and sliding of cellulosic networks by α-expansins (EXPAs) for diffuse growth ^2,3 . Here, we knocked out root-hair specific EXPA7 and EXPA18 in Arabidopsis, abolishing root-hair tip growth which was restored by complementation with genes from some, but not all, expansin clades. Notably, EXPA13 and EXPA20 failed to complement; they belong to two ancient clades lacking a highly conserved Asp considered essential for expansin activity. Mutation of this Asp in EXPA7 confirmed its requirement for wall enlargement. EXPA-mCherry fusions revealed widely contrasting patterns of subcellular trafficking and wall-binding for different EXPAs. The results demonstrate an essential EXPA requirement for root-hair tip growth and uncover a greater diversity of expansin functions than previously recognized.

Plant cell growth depends on slow, irreversible creep of the fibrous cell wall stretched by turgor, yet the mechanics of creep—and how it differs from elasto-plastic deformation—remains uncertain ^1–3 . Using multiscale modeling, we show how wall creep emerges from elementary sliding of cellulose microfibrils along their contact interfaces. A dominant sliding mode involves localized dislocation-like defects that stochastically nucleate at laterally bonded microfibril contacts and then glide along the interface by surmounting discrete energy barriers. Embedding these sliding events into Monte Carlo simulations of stretched cellulose networks recapitulates whole-wall creep kinetics. Elevated stress lowers sliding energy barriers and triggers rapid irreversible deformation characteristic of plastic flow. Compared with elasto-plastic stretching, creep induces less microfibril reorientation but promotes bundling, thereby reducing subsequent yielding. These results clarify the microfibril rearrangements underlying cell wall creep and show how they redistribute stress to permit sustained wall expansion at steady turgor without compromising structural integrity.

Lignification waterproofs and strengthens secondary plant cell walls but increases the energy cost of sugar release for biofuels. The physical association between lignin and the carbohydrate scaffold that accommodates lignin polymerization, along with the distinct roles of lignin units and carbohydrate partners during lignification, remain unclear. Here, we map lignin-carbohydrate spatial proximity by solid-state NMR in ¹³C-labeled Arabidopsis inflorescence stems during secondary cell wall formation. Analyses include wild-type plants and mutants that selectively or globally disrupt lignin biosynthesis. Mature walls in basal regions show enrichment of S-lignin and dense carbohydrate-lignin packing. Acetylated xylan predominantly associates with S-lignin, while methylated pectin unexpectedly interacts with G-lignin during early-stage lignification. The importance of S-lignin in stabilizing the carbohydrate-lignin interface is highlighted by weak lignin-carbohydrate contacts and compromised mechanical properties in the low-S fah1 mutant, whereas the ref3 mutant, despite reduced lignin content, remains unaffected due to a high S/G ratio. Thus, molecular mixing patterns, rather than lignin content, critically determine the structure and properties of lignocellulosic materials.

Plants synthesize polysaccharide-based primary cell walls that possess unique microstructures and mechanical properties to accommodate plant growth and provide protection. It remains challenging to assess the role of polysaccharide organization and interactions in the mechanical behavior of primary cell walls owing to their complex microstructure and highly nonlinear mechanical responses. Employing a coarse-grained molecular dynamics model developed for onion epidermal walls, this work explores the conditions under which polysaccharide assembly and interactions might play a significant role in primary cell wall mechanics. Cellulose-cellulose adhesion plays a dominant role in the wall load-bearing capacity, but when cellulose-cellulose adhesion was disrupted computationally, cellulose-xyloglucan adhesion could influence the wall load-bearing capacity. Contrary to the common concept that xyloglucans mechanically tether well-separated cellulose microfibrils, xyloglucans functioned in this case as interfibrillar adhesives capable of transmitting tensile forces between cellulose microfibrils. Our findings may inform design criteria of new materials inspired by plant cell walls.

Lignification waterproofs and strengthens the secondary plant cell wall, while increasing the energy cost associated with releasing sugars for biofuel production. The physical association between lignin and the carbohydrate scaffold that accommodates lignin polymerization, as well as the temporally distinct roles of different lignin units and carbohydrate partners during lignification, remain largely unclear. Here we map the lignin-carbohydrate interactions by solid-state NMR in ¹³ C-labeled Arabidopsi s inflorescence stems as secondary cell walls are formed. Analysis includes wild-type and two mutants that either selectively or globally disrupt lignin biosynthesis. Mature cell walls in the basal regions of older stems are enriched in S-lignin and carbohydrate-lignin interactions. Acetylated xylan is the dominant mediator of interactions with S-lignin, while methylated pectin unexpectedly interacts with G-lignin during early-stage lignification. The critical role of S-lignin in stabilizing carbohydrate-lignin interface is emphasized by the weak lignin-carbohydrate interactions and compromised mechanical properties of a low-S fah1 mutant, whereas the ref3 mutant, with low overall lignin content but a higher S/G ratio, remained unaffected. These findings demonstrate that the molecular mixing pattern, rather than lignin content, is a key determinant of the structure and properties of lignocellulosic materials.