Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.
Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.
The plant cell wall is a dynamic structure. Growing cells are surrounded by a primary cell wall, the organization of which allows cells to expand. Cells that cease to grow deposit a more rigid secondary wall that enhances the mechanical support of the plant. Both cell walls are composed of cellulose microfibrils embedded in a matrix of polysaccharides of different structures (e.g., hemicellulose and pectin) that vary across the different developmental stage and tissues1,2. Cellulose is synthesized as chains of (1,4)-β-D-glucan that are tightly aligned to form the microfibril of a crystalline structure. Amorphous cellulose refers to the regions where the glucan chains are less ordered. The ratio between the crystalline and the amorphous domains is one parameter thought to affect the mechanical properties of the cell wall, by providing mechanical strength and viscoelastic characteristic, respectively3. Several methods have been developed to detect and quantify the two forms of cellulose arrangement, among them X-ray diffraction and cross polarization/magic angle spinning solid-state NMR4. X-ray diffraction can be used to determine the proportion of crystalline versus amorphous cellulose domains in the sample5. An alternative method uses fractionation of cell wall content into acid-insoluble and acid-soluble material, to distinguish between the crystalline and amorphous cellulose or other polymers, respectively. In this approach, incorporation of labeled glucose ([14C]Glucose) is used to quantify the cellulose6,7. These methods require large volumes of plant material for whole organ analyses, at best, and hence, are inadequately sensitive to tissue-specific variation in cell wall structure. Visualization of cellulose microfibrils at a cellular resolution can be achieved in live imaging studies combined with fluorescent dyes8,9, that can identify changes in the orientation of the cellulose microfibrils. However, these dyes are not used for quantification, they are not specific to crystalline cellulose and may interfere with the normal structure of the cell wall8. Polscope is an imaging technique that relies on the ability of crystalline cellulose to split light beams and retard part of the light10. Light retardation is strongest for microfibrils that lie perpendicularly to the direction of light propagation. For microfibrils with similar orientation, the higher the degree of crystallinity, the larger the light retardance11. Hence, polscope is used to study both the relative levels and orientation of the cellulose microfibrils.
Roots exhibit linear growth, during which cells originating at the stem cell niche, at the tip of the root, undergo a series of cell divisions, before they rapidly expand12. The cells comprising the root expand in a unidirectional (anisotropic) manner, as dictated by small molecule signaling hormones that impact the properties of the cell wall13. Differential responses to hormones, in time and space, provide a means of ensuring balanced organ growth14. Hence, high resolution analysis of cell wall structure can provide important information necessary to better understand the connection between cell type-specific responses to whole organ growth. Here, we report the implementation of polscope to study tissue-specific accumulation of crystalline cellulose in Arabidopsis roots, as observed in high quality anatomical sections. This method recently uncovered cell type-specific accumulation of crystalline cellulose in response to spatial perturbation of hormonal activity15.
1. Plant Growth
2. Fixation
3. Dehydration
4. Infiltration
5. Block Preparation
6. Sectioning
7. Slide Preparation for Polarized Microscope
8. Image Acquisition
9. Polscope Analysis of Cellulose Microfibril Orientation
10. Polscope Analysis of Crystalline Cellulose Accumulation
We study the impact of cell type-specific responses to brassinosteroids (BRs), using the Arabidopsis root as a model organ15-17. When the BR receptor BRI1 is targeted, in the background of bri1 mutant, to a subset of epidermal cells called non-hair cells (Figure 2A,B), it inhibits unidirectional cell expansion of neighboring cells, and whole-root growth15. Polscope analysis was performed to reveal the mechanism underlying this inhibition. Longitudinal sections of the root obtained from wild type and from lines expressing BRI1 in non-hair cells only, showed similar orientation of cellulose microfibrils (Figure 2C,D,and as explained in 9.2.3), enabling comparison of the relative accumulation of crystalline cellulose in meristematic and elongating cells, using cross sections taken from these zones (Figure 2E,F). This analysis showed a correlation between BRI1 expression in non-hair cells and local accumulation of crystalline cellulose. Follow up experiments showed that mild inhibition of cellulose synthase by low concentrations of isoxaben lowered the level of crystalline cellulose in these cells, which in turn, partially restored growth inhibition, by promoting unidirectional cell expansion and root length15.
Figure 1: Polarized Light Microscopy System. The Polscope system consists of a light microscope (A), equipped with a CCD digital camera (B), analyzer and a green liquid crystal located on top of its light source (C). (D) Abrio software was used for image acquisition and analysis. Please click here to view a larger version of this figure.
Figure 2: Polscope Analysis of Cell-Wall Composition. (A) Cross-section of the Arabidopsis primary root showing radial organization of its constituent tissues. Of these, the epidermal non-hair cells (N); hair cells (H) and cortex (C) are marked. Bar, 10 µm. (B) Confocal microscopy images of roots expressing BRI1-GFP targeted to non-hair cells. White, PI staining that marks the cells, Green, BRI1-GFP expression. Bar, 20 µm. (C) Longitudinal sections of roots obtained from wild type and from plants expressing BRI1 in non-hair cells. The angle of cellulose microfibrils (i.e., the angle between the color and cell long axis, reflecting the long axis of the root) is similar between the two lines. The cell wall flanking the cell along its long axis is encircled; angle mark represent the shaded cell wall which is measured. Bar, 50 µm. (D) Quantification of cellulose microfibril angle in epidermal cell walls. Note the high variability of the angles present in meristematic cells (i.e., meristematic zone, MZ) as compared to cells in the transition zone (TZ), as reflected by the box plot. The similar average cellulose microfibril angle between wild type and plants expressing BRI1 in non-hair cells enables comparison of crystalline cellulose accumulation in cells of their corresponding developmental zone. The average angle in each sample is indicated by a red line. (E-F) Transverse root sections of these same plant backgrounds, shown in light retardance mode. Color scale represents light retardance of 0 – 17 nm. Sections corresponding to the meristem (E) and elongation (F) zones are shown. Bar, 50 µm. The outer epidermal cell wall and inner cortical cell wall are encircled. (G) Quantification of the retardance values as calculated from the polscope images. Values are expressed as the ratio of retardance between the outer epidermal cell wall and the inner cortical cell wall. Note that the high deposition of crystalline cellulose in non-hair cells in lines expressing BRI1 in these cells only. Mean +SE; 40 < n < 600; (**) P < 0.01; (***) P < 0.001 in a two-tailed t-test. The figure was adopted and modified from15. Please click here to view a larger version of this figure.
Here, we present a method for determination of the accumulation of crystalline cellulose in the different tissues composing the Arabidopsis roots, while maintaining anatomical information. As such, it provides an additional step towards understanding growth processes in plants at a cellular resolution. This method can be also applied for the study of additional plant species and organs.
A number of points must be considered when applying the method. First, crystalline cellulose accumulation in a given root section can be compared among tissues, among sections from different genotypes and among treatments if the orientation of their cellulose microfibrils is similar. Cellulose microfibrils orientation is determined in longitudinal section containing one face of the cell wall18. Hence, the series of longitudinal sections, covering the distinct tissues of the roots, from the outer to the inner most ones, enable analysis of the cellulose microfibrils orientation in the different tissues, at a given developmental stage. In the example shown here, the meristematic cells of the epidermis have a wide range of cellulose microfibrils orientations with an average cellulose microfibrils angle of 140°, while the elongating cells of the epidermis have a more organized structure, with an average cellulose microfibrils angle of 120°. These measurements were similar between wild type and the mutant of interest, thereby allowing a confident comparison of their relative crystalline cellulose accumulation, in the corresponding cell types and developmental zone.
Secondly, variability in the width of the different sections can impact the levels of light retardance measured. This is circumvented here by normalizing the raw retardance measurements of the tissue of interest (epidermal cells) to the retardance measurement of a different tissue (cortex cells). The normalized values are then compared (See Figure 2E-G).
Finally, transverse sections along the root, capture different developmental zones. Identification of these zones in the cross section is important for interpretation of the data. Loss of the outermost lateral root cap cell layer in the section comprise a straightforward zone marker. In general, cells start their fast expansion when the oldest cell of the lateral root cap undergoes programmed cell death, after which, the unprotected epidermis becomes the outermost tissue19.
Currently, methods that provide a cellular resolution measure of the ratio of the crystalline versus the amorphous cellulose domains are lacking. Indeed, this is also one limitation of the method presented here. However, its ability to communicate the accumulation of crystalline cellulose per se is a significant advance, as high relative levels of crystalline cellulose likely renders a more robust cell wall with increased tensile strength. Development of high resolution tools to precisely determine the composition the cell wall alongside its mechanical properties remains a future challenge.
The authors have nothing to disclose.
We thank Dr. M. Rosenberg for her advice and help with anatomical sectioning. We also thank D. Eisler for his technical assistance. This research was supported by grants from FP7-PEOPLE-IRG-2008, Binational Agriculture research and Development (BARD; IS-4246-09), and Israel Science Foundation (ISF; 592/13).
Murashige & Skoog (MS) | Duchefa | M0221 | |
Borax | LOBA CHEMIE | 6038 | |
Methylene blue | Sigma | M-9140 | |
Azure | Sigma | 861065 | |
Syringe Driven 0.22mm PVDF Filter | MILLEX-GV | ||
Glutaraldehyde | EMS | 16220 | |
Sodium Cacodylate | Sigma | C-0250 | |
Leica kit historesin | Leica | 7022 18 500 | |
embedding molds | Agar Scientific | AGG3530 | |
film 100µ P.P.C. | www.Jolybar.com | overhead film | |
Knivemaker | LKB | 7800 | |
Ultratome III | LKB | 8800 | Ultratome |
Shandon immumount | Thermo | 9990402 | mounting medium |
light microscope | Nikon | Eclipse 80i | |
Abrio imaging system | CRI | Abrio imaging system | |
Abrio V2.2 software | CRI | Abrio V2.2 software | |
Open access polarized light image analysis software | OPS | OpenPolScope | http://www.openpolscope.org/ |