MESOSCALE STRUCTRUAL ANALYSIS OF CELLUOLOSE MICROFIBRILS IN PLANT CELL WALLS USING VIBRATIONAL SUM FREUQNENCY GENERATION (SFG) SPECTROSCOPY

Restricted (Penn State Only)
Author:
Huang, Shixin
Graduate Program:
Chemical Engineering
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
August 01, 2018
Committee Members:
  • Seong Han Kim, Dissertation Advisor
  • Seong Han Kim, Committee Chair
  • Daniel J Cosgrove, Committee Member
  • Phillip E Savage, Committee Member
  • Charles T Anderson, Outside Member
  • Enrique Daniel Gomez, Committee Member
Keywords:
  • Cellulose
  • sum frequency generation
  • plant cell walls
Abstract:
Cellulose, along with matrix polymers and some structural proteins, make up the three-dimensional architecture of plant cell walls. In plant cell walls, cellulose is formed in a fibrillar structure referred to as cellulose microfibril (CMF), interspersed in an amorphous matrix consisting of pectin, hemicelluloses, and lignin. The assembly of the CMFs and the matrix components and the interactions between these determine the mechanical properties of the plant cell walls. Although tremendous progresses have been made in understanding the nanoscale structure of cellulose and microfibrils in plant cell walls, two important questions remain: (1) What are the structure and physical states of cellulose in microfibrils in native plant cell walls, rather than of purified cellulose crystals? (2) What are the mesoscale (⁓100 nm – 1 µm) arrangements of CMFs (bundling feature, interfibrillar distance value, orientation, and packing pattern) in intact plant cell walls? As a non-linear optical technique, sum frequency generation (SFG) vibrational spectroscopy can selectively detect crystalline cellulose without spectral interference from amorphous polymers. The utilization of SFG reveals information of the crystal structures and the mesoscale structural ordering of crystalline cellulose in plant cell walls with minimal pretreatments. A recent important improvement in the SFG study of plant cell walls is to combine the broadband SFG spectroscopic system with an optical microscope to reduce the spatial resolution from >150 µm to <5 µm. Combined with an automated imaging system, this SFG microscopy facilitates studies of inhomogeneous distributions of cellulose microfibrils within single cell walls such us onion epidermal walls as well as among various types of cell walls in plant tissue sections such as Arabidopsis inflorescence stems and bamboo culms. The primary cell walls (PCWs) of land plants are typically hydrated and have rich pectin content; they shrink significantly when the plants become dehydrated. Nonetheless, to circumvent the problems associated with the presence of water, common IR and XRD analyses of cellulose in PCWs are usually performed in the dehydrated state. Our SFG study revealed reversible changes in spectral features upon dehydration and rehydration of onion epidermal walls. Combined with analyses of atomic force microscopy (AFM) and scanning electron microscopy (SEM) imaging and indentation modulus data, such changes could be attributed to local strains in CMFs due to the collapse of the pectin matrix upon dehydration. The importance of studying cellulose structures in the native fully-hydrated state of primary cell walls is recognized from these results. In nature, cellulose has two crystalline polymorphic structures (Iα and Iβ), but the synthesis mechanisms of the two structures are not well understood. Alga Micrasterias has isolated “rosette”-shaped terminal complextes (TCs) in the primary cell wall and hexagonal arrays of “rosette”-shaped TCs in the secondary cell wall, which is different from most algal species that have rectangular- or linear-shaped TCs based on the reported freeze-fracture EM images. From our SFG and infrared spectroscopic analyses, it was found Micrasterias cells produce cellulose Iβ rather than a mixture of larger amount of Iα and smaller amount of Iβ observed in most algal species and bacteria. Some correlations could be seen between the polymorphic structure and crystal size of cellulose and the shape and size of TC, and this information might be used as a basis set needed to understand what determines the cellulose structure and crystallinity in plant cell walls. Mutations on plant may affect the morphological features, content, ordering and orientation of cellulose microfibrils in plant cell walls. Our SFG study found that mutations of primary and secondary cellulose synthases (CESAs) of moss Physcomitrella Paten have negative effects on secondary cell wall cellulose deposition and reduced cellulose content in leaf cell walls. In the study of the transdifferentiated cell walls of Arabidopsis seedlings, SFG spectra of cellulose in transdifferentiated cell walls showed similar spectral features to the cellulose in secondary cell walls of Arabidopsis stem, suggesting the formation of secondary cell walls in the transdifferentiated cells. Comparing the microfibril assembly in Arabidopsis seedling cell walls of pectin mutants to the wild-type, features of SFG spectra indicate that microfibrils have less ordered lateral packing in QUASIMODO2 (QUA2) and TUMOROUS SHOOT DEVELOPMENT2 (TSD2) mutant cell walls.