Molecular insights into the complex mechanics of plant epidermal cell walls.

Plants have evolved complex nanofibril-based cell walls to meet diverse biological and physical constraints. How strength and extensibility emerge from the nanoscale-to-mesoscale organization of growing cell walls has long been unresolved. We sought to clarify the mechanical roles of cellulose and matrix polysaccharides by developing a coarse-grained model based on polymer physics that recapitulates aspects of assembly and tensile mechanics of epidermal cell walls. Simple noncovalent binding interactions in the model generate bundled cellulose networks resembling that of primary cell walls and possessing stress-dependent elasticity, stiffening, and plasticity beyond a yield threshold. Plasticity originates from fibril-fibril sliding in aligned cellulose networks. This physical model provides quantitative insight into fundamental questions of plant mechanobiology and reveals design principles of biomaterials that combine stiffness with yielding and extensibility.

Distinguishing Mesoscale Polar Order (Unidirectional vs Bidirectional) of Cellulose Microfibrils in Plant Cell Walls Using Sum Frequency Generation Spectroscopy.

Cellulose in plant cell walls are synthesized as crystalline microfibrils with diameters of 3-4 nm and lengths of around 1-10 µm. These microfibrils are known to be the backbone of cell walls, and their multiscale three-dimensional organization plays a critical role in cell wall functions including plant growth and recalcitrance to degradation. The mesoscale organization of microfibrils over a 1-100 nm range in cell walls is challenging to resolve because most characterization techniques investigating this length scale suffer from low spatial resolution, sample preparation artifacts, or inaccessibility of specific cell types. Here, we report a sum frequency generation (SFG) study determining the mesoscale polarity of cellulose microfibrils in intact plant cell walls. SFG is a nonlinear optical spectroscopy technique sensitive to the molecular-to-mesoscale order of noncentrosymmetric domains in amorphous matrices. However, the quantitative theoretical model to unravel the effect of polarity in packing of noncentrosymmetric domains on SFG spectral features has remained unresolved. In this work, we show how the phase synchronization principle of the SFG process is used to predict the relative intensities of vibrational modes with different polar angles from the noncentrosymmetric domain axis. Applying this model calculation for the first time and employing SFG microscopy, we found that cellulose microfibrils in certain xylem cell walls are deposited unidirectionally (or biased in one direction) instead of the bidirectional polarity which was believed to be dominant in plant cell walls from volume-averaged characterizations of macroscopic samples. With this advancement in SFG analysis, one can now determine the relative polarity of noncentrosymmetric domains such as crystalline biopolymers interspersed in amorphous polymer matrices, which will open opportunities to study new questions that have not been conceived in the past.

Nanoscale movements of cellulose microfibrils in primary cell walls.

The growing plant cell wall is commonly considered to be a fibre-reinforced structure whose strength, extensibility and anisotropy depend on the orientation of crystalline cellulose microfibrils, their bonding to the polysaccharide matrix and matrix viscoelasticity1-4. Structural reinforcement of the wall by stiff cellulose microfibrils is central to contemporary models of plant growth, mechanics and meristem dynamics4-12. Although passive microfibril reorientation during wall extension has been inferred from theory and from bulk measurements13-15, nanometre-scale movements of individual microfibrils have not been directly observed. Here we combined nanometre-scale imaging of wet cell walls by atomic force microscopy (AFM) with a stretching device and endoglucanase treatment that induces wall stress relaxation and creep, mimicking wall behaviours during cell growth. Microfibril movements during forced mechanical extensions differ from those during creep of the enzymatically loosened wall. In addition to passive angular reorientation, we observed a diverse repertoire of microfibril movements that reveal the spatial scale of molecular connections between microfibrils. Our results show that wall loosening alters microfibril connectivity, enabling microfibril dynamics not seen during mechanical stretch. These insights into microfibril movements and connectivities need to be incorporated into refined models of plant cell wall structure, growth and morphogenesis.