Progress and Opportunities in the Characterization of Cellulose - An Important Regulator of Cell Wall Growth and Mechanics

Abstract

The plant cell wall is a dynamic network of several biopolymers and structural proteins including cellulose, pectin, hemicellulose and lignin. Cellulose is one of the main load bearing components of this complex, heterogeneous structure, and in this way, is an important regulator of cell wall growth and mechanics. Glucan chains of cellulose aggregate via hydrogen bonds and van der Waals forces to form long thread-like crystalline structures called cellulose microfibrils. The shape, size, and crystallinity of these microfibrils are important structural parameters that influence mechanical properties of the cell wall and these parameters are likely important determinants of cell wall digestibility for biofuel conversion. Cellulose-cellulose and cellulose-matrix interactions also contribute to the regulation of the mechanics and growth of the cell wall. As a consequence, much emphasis has been placed on extracting valuable structural details about cell wall components from several techniques, either individually or in combination, including diffraction/scattering, microscopy, and spectroscopy. In this review, we describe efforts to characterize the organization of cellulose in plant cell walls. X-ray scattering reveals the size and orientation of microfibrils; diffraction reveals unit lattice parameters and crystallinity. The presence of different cell wall components, their physical and chemical states, and their alignment and orientation have been identified by Infrared, Raman, Nuclear Magnetic Resonance, and Sum Frequency Generation spectroscopy. Direct visualization of cell wall components, their network-like structure, and interactions between different components has also been made possible through a host of microscopic imaging techniques including scanning electron microscopy, transmission electron microscopy, and atomic force microscopy. This review highlights advantages and limitations of different analytical techniques for characterizing cellulose structure and its interaction with other wall polymers. We also delineate emerging opportunities for future developments of structural characterization tools and multi-modal analyses of cellulose and plant cell walls. Ultimately, elucidation of the structure of plant cell walls across multiple length scales will be imperative for establishing structure-property relationships to link cell wall structure to control of growth and mechanics.

Keywords: X-ray diffraction; X-ray scattering; atomic force microscopy; cellulose allomorphs; cellulose crystallinity; cellulose microfibrils; nuclear magnetic resonance spectroscopy; vibrational spectroscopy.

FIGURE 1

Tools enabling characterization of the primary cell wall at different length scales. Crystal structures of cellulose Iα and Iβ are reprinted with permission from Nishiyama et al. (2003). Crystal Structure and Hydrogen Bonding System in Cellulose Iα from Synchrotron X-ray and Neutron Fiber Diffraction. Journal of the American Chemical Society 125, 14300–14306. Copyright 2003 American Chemical Society. Primary cell wall is reprinted with permission from Cosgrove (2014). Re-constructing our models of cellulose and primary cell wall assembly. Current Opinion in Plant Biology 22, 122–131. Copyright © 2014 Elsevier Ltd. Schematic inspired by Martínez-Sanz et al. (2015a). Application of X-ray and neutron small angle scattering techniques to study the hierarchical structure of plant cell walls: A review. Carbohydrate Polymers 125, 120–134.

FIGURE 2

Synchrotron XRD 2D pattern of Halocynthia cellulose Iβ. Reprinted with permission from Nishiyama et al. (2002). Crystal Structure and Hydrogen-Bonding System in Cellulose Iβ from Synchrotron X-ray and Neutron Fiber Diffraction. Journal of the American Chemical Society 124, 9074–9082. Copyright © 2002, American Chemical Society.

FIGURE 3

Chain packing in unit cell of cellulose Iα (left) and cellulose Iβ (right). Reproduced from Koyama et al. (1997). Parallel-Up Structure Evidences the Molecular Directionality during Biosynthesis of Bacterial Cellulose PNAS 94 (17), 9091–9095. Copyright © 1997, The National Academy of Sciences of the United States.

FIGURE 4

Techniques used to characterize cellulose polymorphism: (A) XRD, (B) NMR, (C) SFG, (D) IR, and (E) Raman. Reprinted by permission from RightsLink Permissions: Springer Nature. Cellulose. Cellulose polymorphism study with sum-frequency-generation (SFG) vibration spectroscopy: identification of exocyclic CH2OH conformation and chain orientation, Lee, C. M., Mittal, A., Barnette, A. L., Kafle, K., Park, Y. B., Shin, H., Johnson, D. K., Park, S., and Kim, S. H., Copyright © 2013.

FIGURE 5

X-ray diffraction methods for determination of the crystallinity of cellulose. Reprinted by permission from RightsLink Permissions: Springer Nature. Cellulose. Comparison of sample crystallinity determination methods by X-ray diffraction for challenging cellulose I materials, Ahvenainen, P., Kontro, I., and Svedström, K., Copyright © 2016.

FIGURE 6

Transmission Electron Microscopy micrographs comparing the morphology of cellulose microfibrils isolated by different chemical treatments. (a) Peroxide alkaline, (b) peroxide-alkaline-hydrochloric acid, (c) 5 wt% potassium hydroxide, and (d) 18 wt% potassium hydroxide. The combination of peroxide alkaline and hydrochloric acid or the application of a high concentration (18 wt%) potassium hydroxide solution leads to shorter microfibrils, suggesting these treatments can cause microfibril scission. Reprinted from Carbohydrate Polymers, 76, Zuluaga, R., Putaux, J. L., Cruz, J., Vélez, J., Mondragon, I., Gañán, P. Cellulose microfibrils from banana rachis: Effect of alkaline treatments on structural and morphological features, 51–59, Copyright © 2009, with permission from Elsevier.

FIGURE 7

Atomic Force Microscopy micrograph of cellulose microfibrils merging in and out of microfibril bundles. Reprinted with permission from Zhang et al. (2016). Spatial organization of cellulose microfibrils and matrix polysaccharides in primary plant cell walls as imaged by multichannel atomic force microscopy. The Plant Journal 85, 179–192. Copyright © 2016, John Wiley and Sons.

FIGURE 8

Confocal microscopy images showing (A) cellulose orientation in different cell wall layers using S4B staining, and (B) rotation of stained microfibrils over time in Arabidopsis. Reprinted with permission from Anderson et al. (2010). Real-Time Imaging of Cellulose Reorientation during Cell Wall Expansion in Arabidopsis Roots. Plant Physiology 152 (2), 787–796. www.plantphysiol.org. Copyright © 2010 American Society of Plant Biologists.

FIGURE 9

Field emission scanning electron microscopy (FESEM) images of onion cell wall without enzyme treatment (a,a’) and after different enzyme treatments (b–d,b’–d’). (e–g) Effect of treatments on wall properties. XGase is a family-12 glycosyl hydrolase (GH) that hydrolyzes xyloglucan but not unbranched glucan, Case is a family-12 GH that hydrolyzes non-crystalline cellulose only, and CXGase is a family-5 GH that hydrolyzes both non-crystalline cellulose and xyloglucan. No EG denotes no endoglucanase treatment. Reprinted with permission from Zheng et al. (2018). Xyloglucan in the primary cell wall: assessment by FESEM, selective enzyme digestions and nanogold affinity tags. The Plant Journal 93, 211–226. Copyright 2018, John Wiley and Sons.