title: Electron tomography of cryo-immobilized plant tissue: a novel approach to studying 3D macromolecular architecture of mature plant cell walls in situ.
abstract: Cost-effective production of lignocellulosic biofuel requires efficient breakdown of cell walls present in plant biomass to retrieve the wall polysaccharides for fermentation. In-depth knowledge of plant cell wall composition is therefore essential for improving the fuel production process. The precise spatial three-dimensional (3D) organization of cellulose, hemicellulose, pectin and lignin within plant cell walls remains unclear to date since the microscopy techniques used so far have been limited to two-dimensional, topographic or low-resolution imaging, or required isolation or chemical extraction of the cell walls. In this paper we demonstrate that by cryo-immobilizing fresh tissue, then either cryo-sectioning or freeze-substituting and resin embedding, followed by cryo- or room temperature (RT) electron tomography, respectively, we can visualize previously unseen details of plant cell wall architecture in 3D, at macromolecular resolution (∼ 2 nm), and in near-native state. Qualitative and quantitative analyses showed that wall organization of cryo-immobilized samples were preserved remarkably better than conventionally prepared samples that suffer substantial extraction. Lignin-less primary cell walls were well preserved in both self-pressurized rapidly frozen (SPRF), cryo-sectioned samples as well as high-pressure frozen, freeze-substituted and resin embedded (HPF-FS-resin) samples. Lignin-rich secondary cell walls appeared featureless in HPF-FS-resin sections presumably due to poor stain penetration, but their macromolecular features could be visualized in unprecedented details in our cryo-sections. While cryo-tomography of vitreous tissue sections is currently proving to be instrumental in developing 3D models of lignin-rich secondary cell walls, here we confirm that the technically easier method of RT-tomography of HPF-FS-resin sections could be used immediately for routine study of low-lignin cell walls. As a proof of principle, we characterized the primary cell walls of a mutant (cob-6) and wild type Arabidopsis hypocotyl parenchyma cells by RT-tomography of HPF-FS-resin sections, and detected a small but significant difference in spatial organization of cellulose microfibrils in the mutant walls.
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, California, United States of America; Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Purbasha
authors.initials: P
authors.lastname: Sarkar
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, California, United States of America; Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Elena
authors.initials: E
authors.lastname: Bosneaga
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Edgar G
authors.initials: EG
authors.lastname: Yap
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, California, United States of America.
authors.forename: Jyotirmoy
authors.initials: J
authors.lastname: Das
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Wen-Ting
authors.initials: WT
authors.lastname: Tsai
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, California, United States of America.
authors.forename: Angelo
authors.initials: A
authors.lastname: Cabal
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Erica
authors.initials: E
authors.lastname: Neuhaus
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, California, United States of America.
authors.forename: Dolonchampa
authors.initials: D
authors.lastname: Maji
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, California, United States of America.
authors.forename: Shailabh
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Michael
authors.initials: M
authors.lastname: Joo
authors.affiliation: Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Sergey
authors.initials: S
authors.lastname: Yakovlev
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Roseann
authors.initials: R
authors.lastname: Csencsits
authors.affiliation: Department of Computer Science, University of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: Zeyun
authors.initials: Z
authors.lastname: Yu
authors.affiliation: Department of Computer Sciences & The Institute of Computational Engineering and Sciences, University of Texas, Austin, Texas, United States of America.
authors.forename: Chandrajit
authors.initials: C
authors.lastname: Bajaj
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Kenneth H
authors.initials: KH
authors.lastname: Downing
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, California, United States of America; Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Manfred
authors.initials: M
authors.lastname: Auer
mesh_terms: D017360:Arabidopsis / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*; D018914:Cells, Immobilized / Q000648:ultrastructure; D002482:Cellulose / Q000378:metabolism; D015925:Cryopreservation; D016616:Cryoultramicrotomy; D055032:Electron Microscope Tomography / Q000379:methods*; D017110:Freeze Substitution; D021621:Imaging, Three-Dimensional / Q000379:methods*; D009154:Mutation; D013696:Temperature; D016610:Tissue Embedding
chemical_list: D002482:Cellulose