From Structure to Dynamics: Direct Visualization of Primary Plant Cell Walls by Atomic Force Microscopy

Open Access
Author:
Zhang, Tian
Graduate Program:
Cell and Developmental Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
March 03, 2015
Committee Members:
  • Daniel J Cosgrove, Dissertation Advisor
  • Daniel J Cosgrove, Committee Chair
  • Sarah Mary Assmann, Committee Member
  • Ming Tien, Committee Member
  • William O Hancock, Committee Member
Keywords:
  • plant cell walls
  • AFM
  • dynamics
  • biomechanics
  • loosening
  • growth
Abstract:
Macroscale properties of plant cell walls originate from the nano scale interactions beween microfibrils and matrix polymers, the details of which have not been settled, hampering our understanding of how the plant cell wall enables loosening during growth while maintaining its mechanical strength. To visualize the spatial arrangement of the most recently deposited cell wall components at the nm scale, we used multi-channel atomic force microscopy (AFM) in PeakForce Tapping® mode to map variations in the surface height, modulus and tip adhesion across the innermost surface of never-dried primary onion epidermal walls. Height maps and PeakForceTM error maps selectively visualized microfibrils and obscured the matrix polymers in hydrated primary walls whereas nanomechanical mapping reveals significant polymer heterogeneity in stiffness and adhesiveness at the nm scale. By selectively merging these maps, distribution of soft matrix polymers and stiffer microfibrils became evident. Individual microfibrils frequently emerged into and out of short regions of microfibril bundles, resembling the previously predicted structure of ‘biomechanical hot spots’, the load bearing sites for cell wall loosening. By time lapse AFM imaging of relaxed cell walls, microfibril dynamics (motion) were seen to increase upon incubation with Cel12A, an endoglucanase capable of inducing cell wall loosening, whereas xyloglucanase and EDTA had no effect. This confirms the role of Cel12A sites as biomechanical hot spots that stabilize and reinforce the microfibril network. By monitoring the nano scale microfibril motions during cell wall extension, we observed that microfibrils reoriented and compressed transversely during physical uniaxial extension, whereas they separated during Cel12A-induced creep. Physical extension also caused kinking of microfibrils, which was reduced by Cel12A treatment. Microfibril sliding as well as shearing was also observed during physical extension and Cel12A-induced creep. These results provided molecular basis for cell wall extension and loosening, suggesting that microfibrils are connected at limited regions, dubbed ‘biomechanical hot spots’ which are targeted by Cel12A in order for microfibrils to decouple and separate during creep. Nanomechanical mapping detected increases in modulus of microfibrils upon extension, due to tensioning of the microfibrils; the modulus (and tension) reduced after Cel12A treatment.