Scanning microdiffraction of longitudinal sections of Arabidopsis stem
Scanning X-ray microdiffraction of longitudinal sections of stems from wild type and seven mutants of Arabidopsis was carried out in order to assess fibril orientation and organization in the tissues of the stem. Approximately, 16,000 diffraction patterns were collected on 32 samples. Samples were harvested and stems microtomed into 100-micron-thick longitudinal sections and stored in water (either 3 days or 30 days) until just prior to data collection at which point they were dried in air for 24 h. Analysis of two repeats of fresh and stored samples resulted in essentially identical results in experiments carried out over 2 years.
A 5-micron X-ray beam was scanned over a grid with 5-micron step size, and a diffraction pattern was collected at each grid point. The scattering angles subtended by the patterns ranged from a spacing of ~100 Å at the beam stop (small-angle regime) to ~2 Å at the detector edge (wide-angle regime). In most cases, a 300 mm sample to detector distance was used. Figure 1 provides an example of data collected from a thin section of wild-type Arabidopsis stem. Samples were aligned using a coaxial optical microscope with a hole down the optical axis to accommodate the X-ray beam thereby allowing precision alignment of the beam to select positions on the sample. Figure 1a shows an optical micrograph of the wild-type sample with a blue rectangle marking the position of a 160 × 3 grid of positions from which diffraction patterns were taken. Figure 1c contains 57 diffraction patterns selected to represent data from the cortex, xylem, and the region adjacent to the pith.
Software was developed to automatically extract specific structural information from these patterns (see “Methods” section for details). A number of structural parameters were extracted from each diffraction pattern and their variation as a function of distance across the stem mapped. Figure 1b includes the distribution of fiber content, microfibril angle, and axial coherence across the sample pictured in Fig. 1a. Each of these parameters is described in more detail below. The distributions represent a mapping across the entire breadth of the stem, starting with the epidermis, moving to the vascular tissues and then the pith, and continuing to reverse this sequence to the other side of the stem. The organization of tissues in Arabidopsis stems is not circularly symmetric about the step axis, and within the 100 micron thickness of the sections used, there is an occasional superposition of regions from different tissues. The cambium, the region between xylem and phloem appears to correspond to the region with the greatest fiber content, consistent with our earlier observations [9]. From the mapping of specific properties, representative parameters were extracted to ease the comparison of structural order present in the different variants.
Oriented fiber content
We have observed that the proportion of material exhibiting orientation varies widely among the mutants studied here (as well as among different tissues within individual stems). In order to quantitate this variation, we chose to calculate a measure of the proportion of total material made up of oriented cellulose fibrils. As implied by the results summarized in Fig. 12, not all cellulosic material is well oriented. But the vast majority of oriented scattering can be attributed to cellulose fibrils. Consequently, the ratio of oriented intensity to disoriented intensity provides a relative measure of the extent of structural organization of cellulose fibrils within the scattering volume. As shown in Fig. 1b, oriented fibril content is the largest in the vascular regions, particularly the xylem, and essentially zero in the pith and epidermis where no scattering attributable to oriented, fibrillar cellulose is observed.
Variations of oriented fiber across the stem for each mutant and wild type have been exhibited in Additional file 1: Figures S1–S8. In order to compare the proportion of oriented fibrils in the different variants, we focused on the cambium the region between xylem and phloem which exhibits the highest content of oriented fibrils [9]. Figure 2 shows a bar graph which compares the maximum values of oriented fiber content observed in each of the eight samples (utilization of average values—rather than maximum—results in a plot exhibiting the same trends). Figure 2 also shows that only ccr and ref3-2 exhibit significant reduction of fiber content. ref3-2 and ccr are the samples with reduced lignin content [21] suggesting that assembly of cellulose fibrils into oriented structures parallel to the stem relies on the presence of normal levels of lignin. ccr has high levels of ferulic acid-containing cell walls and is more digestible than wild type.
Crystalline order in the cellulose fibrils
The positions of the (1 1 0)/(1 −1 0) and (2 0 0) reflections of cellulose provide a measure of the lattice dimensions in the directions perpendicular to the fibril axis. The widths of these reflections are determined by a combination of crystallite breadth (number of cellulose chains in the fibril), cross-sectional shape, degree of order in the packing of cellulose chains, and heterogeneity of crystal lattice constants. The difficulty in separating these three variables is a key reason for the continued debate over the number of cellulose molecules making up an elementary fibril. Nevertheless, the sharpness of these peaks provides a measure of the homogeneity of crystalline order within the cellulose fibrils averaged over the scattering volume. Figure 3 shows the traces of the (1 1 0)/(1 −1 0) and (2 0 0) reflections in scattering from the different lignin mutants. Each trace comes from diffraction pattern exhibiting the highest fiber content for corresponding mutants. The pattern corresponding to the highest fiber content from each sample is compared here for the eight samples analyzed 3 days after harvesting. The (2 0 0) reflection is broadest for ccr and ref3-2, the samples with reduced lignin content. Analysis of Raman spectrum showed that either disorganization or size of crystalline cellulose fibril within plant cell could lead to variation of half width of (2 0 0) reflections [22]. Whether this is due to intrinsic disorder within individual fibrils or to a structurally heterogeneous population of fibrils cannot be determined with existing data. However, these also have the lowest crystalline cellulose content suggesting that lowered lignin content leads to both a decrease in the fraction of crystalline cellulose and the degree of order intrinsic to the crystalline cellulose fibrils.
Microfibril angle
X-ray patterns from many cellulose containing plants exhibit a double orientation—they appear as two diffraction patterns superimposed and rotated relative to one another. The explanation for this observation is the helical winding of cellulose about plant cells that lead to different orientations of the cellulose on the near and far side of a cell [23] as diagrammed in Fig. 4. Microfibril angle was calculated by transforming all intensity onto a polar coordinate system and identifying angles of maximum intensity as detailed in “Methods” section. Because of the symmetry of scattering from cellulose, two independent measures of microfibril angle are obtained for each pattern and these were averaged. In many cases, the diffraction was almost circularly symmetric and no microfibril angle could be determined. Figure 1b shows the distribution of microfibril angle for the wild-type sample. Figure 5 shows that the helical winding of cellulose in the cortex and outer part of vasculature tissue has relatively constant microfibril angles of about 15°, but the angle gradually increases across the inner part of the vascular bundles and fibers to about 30° immediately adjacent to the pith. The lower microfibril angle correlated with observation from confocal Raman microscopy is consistent with the observations of Gielinger [24] and Mateu et al. [25] who reported that orientation of cellulose tends to be parallel to the elongated direction at outer xylem and cortex. Orientation was seldom observed in the pith, precluding an estimate microfibril angle. For many of the mutants with severe reductions in lignin content, orientation is so poor as to preclude measurement of microfibril angle. C4H::F5H fah1-2 mutants exhibit a microfibril angle distribution similar to fresh samples. The trends of microfibril angle for other samples are less clear. See Additional file 1 for details.
Microfibril angle tends to be inversely related to oriented fiber content within individual samples. In a comparison of wild-type and the lignin variants, there is no well-defined correlation between microfibril angle and fiber content. Measurement of microfibril angle is in some cases precluded for mutants with severe reduction of lignin content. As shown in Figs. 4 and 5, the microfibril angles display the tilt to longitudinal direction of cell, larger microfibril angle indicates cellulose fibrils were more transversely arranged within the cell wall. For instance, only small portions of the ref3-2 mutant stem exhibit diffraction patterns with the split reflections required to measure microfibril angle. The mean microfibril angle in the xylem of ref3-2 is about 25°, which is greater than that for wild type and C4H::F5H fah1-2 and suggests that the transverse assembly of cellulose is altered in ref3-2.
It had been well established that in the interfascicular or vascular tissues of Arabidopsis stem, the deposition of lignin decreases as one approaches the pith [26]. Decreasing lignin concentration correlates with decreased fiber content and increased microfibril angle, but to what extent there are causal relationships among these three variables is unclear.
Axial coherence length
A key measure of the crystallinity of cellulose is its axial coherence length. In the axial direction, a useful measure of the degree of imperfection is the coherence length as estimated by the breadth of the (0 0 4) reflection in fiber diffraction patterns. Coherence length may vary among the tissues of the stem as detailed in the figures in the Additional file 1. For simplicity, we chose to compare the distribution of axial coherence lengths observed for each of the lignin mutants as shown in the histograms in Fig. 6. Figure 7 provides comparison of the maximum coherence lengths observed for each of the samples. In fresh samples, ref3-2 and samples with high content of aldehydes ([cadc cadd fah1-2] and [cadc cadd]) appear to have a somewhat lower coherence lengths, with fah1-2 and C4H::F5H fah1-2 exhibiting slightly higher average coherence lengths than wild type.
Coherence length of a cellulose fibril may be influenced by the cross-links it makes with other cell wall constituents. Cross-links may not be easily accommodated by a highly regular crystalline structure. This suggests that coherence length might provide a measure of the degree to which other constituents disrupt the regular ordering of fibrils. Our data suggest that interactions of cellulose with fah1-2 and C4H::F5H fah1-2 provide somewhat less disruption of cellulose order than the interactions in wild type, while the degree of crystallinity remains unchanged (Fig. 2). Aldehyde containing lignins have lower coherence length, suggesting potentially greater interactions, again without altering crystallinity. ref3-2, with lower lignin content, might be expected to place fewer constraints on the organization than wild type, but lowered crystallinity could offset that, perhaps leading to the observed lower coherence length. For ccr, the balance between lowered crystallinity and lowered constraints results in no change in observed coherence length.
In principle, we could also calculate the coherence length for the unoriented fraction of cellulose using curves similar to those in Fig. 13. In practice, the (0 0 4) reflections in these traces are weak and broad, making accurate measurement of coherence length difficult, while clearly indicating that the coherence length is significantly less for the unoriented fraction than for the oriented fraction of cellulose. In all likelihood, this reflects the greater curvature of fibrils expected in the unoriented fraction.
Packing of cellulose fibrils
The small angle region of the diffraction patterns collected here provides structural information about features ranging from 25 to 100 Å in size. The intensity distribution in this region corresponds to the scattering from individual cellulose fibrils. When the fibrils are arranged in an organized fashion, regularly spaced side-to-side, the intensity is modulated by an ‘interference function’ that provides information on the spacing of fibrils in the material [7, 8, 27, 28]. Figure 8 shows the intensity distribution in the small angle region of a wild-type sample, an enlargement of the small angle region of exposure 61 and equatorial traces for exposures 15, 30, 45, and 61. The trace through the exposure (taken from a diffraction pattern of a region immediately adjacent to the pith) shows a modulation of the small angle scattering intensity with peak at 1/day ~0.017 Å−1, suggesting that the fibrils are spaced with a nearest neighbor distance of approximately 60 Å. The observation of this interference near the pith is unexpected because this is the region of the stem with the lowest (observable) oriented fibril content. If the region was homogeneous, the spacing observed would imply an oriented fibril content of at least 25 %, far higher than we observe. Therefore, the region must be highly heterogeneous, with the small fraction of oriented fibrils well—ordered in spatially confined regions.
Impact of water storage on molecular structure of plant cell wall
In addition to collecting SXMD data on samples harvested 3 days prior to data collection, we collected SXMD data on samples that were stored in water for 30 days at room temperature prior to data collection. Our observations show that water storage contributes to observable structural variations in a lignin-dependent manner.
Impact on oriented fiber content
Storage reduced the observed fiber content of wild type only slightly and the reduction of fiber content in C4H::F5H fah1-2 and fah1-2 was negligible. However, storage significantly lowered the levels of crystalline cellulose in the high aldehyde samples, cadc cadd, cadc cadd fah1, and med5a med5b ref8 sample even though their fiber content was comparable to wild type in fresh samples (shown by red bar in Fig. 9). These results were replicated on two sets of samples grown, harvested and analyzed independently at different times. These observations suggest that lignin organization may be important for protecting cellulose from degradation that may occur in aqueous environments over time. Other samples exhibited less change on storage. Interestingly, Fig. 9 shows that, although ccr samples stored in water for 3 days exhibit lower fiber content than wild type, ccr does not show a decrease in oriented fiber content after storage. No variant exhibited a significant increase in oriented fibril content over wild type.
Impact on axial coherence length
Figure 10 shows that exposure to water for 30 days results in most cases in a decrease in coherence length. ref3-2 appears to be the exception. It's very low coherence length appears to increase on storage in water—perhaps through the relaxation of cross-linking constraints on its cellulosic structures. Interestingly, there is no direct correlation between the coherence length and fiber content for the lignin mutants studied here. This may be the result of two competing factors—cellulose crystallinity, which should correlate with increased coherence length; and cross-links to other constituents which should correlate with decreased coherence length.