New Techniques Enable Comparative Analysis of Microtubule Orientation, Wall Texture, and Growth Rate in Intact Roots of Arabidopsis

Abstract

This article explores root epidermal cell elongation and its dependence on two structural elements of cells, cortical microtubules and cellulose microfibrils. The recent identification of Arabidopsis morphology mutants with putative cell wall or cytoskeletal defects demands a procedure for examining and comparing wall architecture and microtubule organization patterns in this species. We developed methods to examine cellulose microfibrils by field emission scanning electron microscopy and microtubules by immunofluorescence in essentially intact roots. We were able to compare cellulose microfibril and microtubule alignment patterns at equivalent stages of cell expansion. Field emission scanning electron microscopy revealed that Arabidopsis root epidermal cells have typical dicot primary cell wall structure with prominent transverse cellulose microfibrils embedded in pectic substances. Our analysis showed that microtubules and microfibrils have similar orientation only during the initial phase of elongation growth. Microtubule patterns deviate from a predominantly transverse orientation while cells are still expanding, whereas cellulose microfibrils remain transverse until well after expansion finishes. We also observed microtubule-microfibril alignment discord before cells enter their elongation phase. This study and the new technology it presents provide a starting point for further investigations on the physical properties of cell walls and their mechanisms of assembly.

Disorganization of Cortical Microtubules Stimulates Tangential Expansion and Reduces the Uniformity of Cellulose Microfibril Alignment among Cells in the Root of Arabidopsis

“…Studies like these support the idea that microtubules are required to coordinate the alignment of microfibrils among cells; however the polylamellate condition makes interpreting the experiments difficult. Here, we took advantage of the Arabidopsis root, in which cell walls in the growth zone lack conspicuous lamellation (Zhu et al, 1998) and have consistently transverse microfibrils (Sugimoto et al, 2000), and show that disrupting microtubules can break down the global coherence of microfibril alignment. How can the global alignment among microfibrils be governed by microtubules, short of mystical forces acting at a distance?…”

“…However, most cells may lie between these extremes. In the Arabidopsis root, an intermediate level of microtubule guidance is suggested by several hours elapsing before microfibril alignment changes when microtubules are depolymerized completely (Sugimoto et al, 2003) or when they reorient from transverse to helical (Sugimoto et al, 2000). Microtubules could guide self-assembly by coordinating a membrane-spanning protein scaffold that binds nascent microfibrils and thereby influences their alignment (Baskin, 2001).…”

“…Samples were prepared for FESEM as described by Sugimoto et al (2000), with minor modifications. Samples were fixed in 4% (v/v) formaldehyde, 20 mM PIPES (pH 7.0), and 0.5 mM CaCl 2 for 2 h at room temperature, washed in 20 mM PIPES, and cryo-protected by incubating in 25% (v/v) DMSO in water for 30 min followed by 50% DMSO until cryo-sectioning at 2115°C.…”

Developmentally equivalent tissue sampling based on growth kinematic profiling of Arabidopsis inflorescence stems

“…They allow for fine-scale characterization of primary stem segment REGRs, whereas natural features on the stem surface are typically not present in sufficiently high density, and do not present precise proxy to cohorts of cells. Similarly, the arbitrary placement of synthetic marks such as graphite particles on roots (Erickson & Sax, 1956;Mullen et al, 1998;Sugimoto et al, 2000) or silicone beads on trichomes (Schwab et al, 2003) substantially complicates the tracking and harvesting of the marked segments.…”

A Comparison of Atomic Force Microscopy and Field-Emission Scanning Electron Microscopy for Imaging the Plant Cell Wall

“…A common way to examine cell wall ultrastructure has been where the surface is exposed and a metal-carbon replica made and examined with transmission electron microscopy. More recently, cell wall surfaces have been imaged directly with field-emission scanning electron microscopy (FESEM), which removes the difficulty of handling a replica [1,2]. However, FESEM requires the sample be dehydrated and critically point-dried, which may alter structure, and a metal coat is usually needed to generate sufficient contrast and avoid charging.…”