A Raman-scattering Study on the Net Orientation of Biomacromolecules in the Outer Epidermal Walls of Mature Wheat Stems (Triticum aestivum)

Abstract

• Background and Aims Raman spectroscopy can be used to examine the orientation of biomacromolecules using relatively thick samples of material, whereas more traditional means of analysing molecular structure require prior isolation of the components, which often destroys morphological features. In this study, Raman spectroscopy was used to examine the outer epidermal cell walls of wheat stems.

• Methods Polarized Raman spectra from the epidermal cell walls of wheat stem were obtained using near-infrared–Fourier transform Raman scattering. By comparing spectra taken with Raman light polarized perpendicular or parallel to the longitudinal axis of the cell, the orientation of macromolecules in the cell wall was investigated.

• Key Results The net orientation of macromolecules varies in the epidermal cell walls of the different components of wheat stem. The net orientation of cellulose is parallel to the longitudinal axis of the cells, whereas the xylan and the phenylpropane units of lignin tend to lie perpendicular to the longitudinal axis of the cells, i.e. perpendicular to the net orientation of cellulose in the epidermal cell walls.

• Conclusions The results imply that cellulose, lignin and xylan form a relatively ordered network that defines the mechanical and structural properties of the cell wall. Such results are likely to have a significant impact on the formulation of definitive models for the static and growing cell wall.

INTRODUCTION

Any mature plant cell wall consists of three types of molecule: two polysaccharides (cellulose and hemicellulose) and a polymer of phenolic compounds (lignin) (Fry, 2001). Cellulose is a homopolymer of β-[1,4] d-glucose molecules linked in a linear chain, with alternating sub-units in crystalline cellulose rotated through 180° (Marchessault and Sundararajan, 1983; Read and Bacic, 2002). Hemicellulose is a natural polymer that is built not only from glucose monomers but also from other monosaccharides such as xylose, mannose, arabinose and galactose. It forms a branched skeleton with an amorphous structure that adsorbs water much more easily than does cellulose (Morrison, 2001). Lignin is a complex polymer of phenylpropane units. It is formed by dehydrogenative polymerization, promoted by peroxidase in the presence of hydrogen peroxide, of three alcohols: p-coumaryl (trans-3-(4-hydroxyphenyl)-2-propen-1-ol), coniferyl (trans-3-(3-methoxy-4-hydroxyphenyl)-2-propen-1-ol) and sinapyl (trans-3-(3,5-dimethoxy-4-hydroxyphenyl)-2-propen-1-ol) (Helm, 2000; Donaldson, 2001). Although the main chemical structures of plant cell-wall constituents are well characterized, their molecular organization within native tissue, with the exception of cellulose, is still poorly understood (Sugimoto et al., 2000; Verbelen and Kerstens, 2000; Read and Bacic, 2002; Kerstens and Verbelen, 2003).

Commonly used methods for investigating morphological features are electron microscopy, optical microscopy and polarization confocal microscopy, which are limited in their ability to reveal information regarding molecular structure. However, traditional means of analysing molecular structure require prior isolation of the various components, which often destroys the morphological features of the cell-wall components (Atalla and Agarwal, 1985; Durot et al., 2003; Fromm et al., 2003).

The classical way of determining the orientation of cellulose in the cell wall is by polarized light microscopy and Fourier transform infrared spectroscopy (FTIR). These methods are limited to very thin preparations containing only one cell layer and cannot be used for preparations containing many cell layers or whole plant organs. IR and Raman spectroscopy have provided details regarding the structure of plant materials (Edwards et al., 1997; Chen et al., 1998; Mouille et al., 2003; Cao et al., 2004). Raman spectroscopy is preferred for biomaterials. This is because Raman scattering occurs only at very low probability so even very thick samples can be studied. In addition, Raman scattering is essentially unaffected by water because water is a weak Raman scatterer. The backbone bonds of cell-wall biomacromolecules, however, are highly birefringent, but they do not have large dipole moments owing to the repetition of identical units, which cancels adjacent dipoles. By contrast, many of the side groups have large moments. Therefore, the Raman intensities associated with the backbone vibration are often intense, whereas the IR intensities are weak, and vice versa for side vibrations. Because many of the important properties of biopolymers are controlled by the orientation of the polymer backbone, Raman spectroscopy provides more information about the polymer than does IR spectroscopy. However, conventional Raman spectroscopy using a visible excitation laser produces a fluorescent emission that interferes with the Raman measurement and can cause sample damage as a result of the high energy of the visible laser used. Using a near-infrared (NIR) excitation laser dramatically reduces these problems (Ma and Phillips, 2002). Therefore, the variety of samples from living tissue that could be investigated was increased, and no complicated procedures were required to obtain near-infrared Fourier transform Raman (NIR-FT Raman) spectra of lignocellulosic materials (Schrader et al., 1999).

In this present work, the NIR-FT Raman spectroscopic technique was used to determine the orientation of biomacromolecules in the outer epidermal cell walls of the different components of mature wheat stem: stalk, node and sheath.

MATERIALS AND METHODS

Plant material

Mature wheat stems (Triticum aestivum) were washed thoroughly with tap water until the washings were clean and colourless and these were then gently freeze-dried. The dried samples were de-waxed by extraction with toluene/ethanol (2 : 1, v/v) in a Soxhlet apparatus for 6 h, after which they were freeze-dried.

Spectroscopic measurements

A Bruker IFS 100 FT-Raman spectrometer equipped with a Ge detector was used to obtain the FT-Raman spectra. NIR excitation at 1064 nm was provided by a Nd:YAG laser. The spectra were measured by averaging 1024 scans at an 8-cm⁻¹ resolution, and the laser power at the sample was set at 400 mW. A single polarizer was inserted into the path of the Raman beam between the sample and the detector. For reproducibility, five specimens were tested from every organ of wheat stem and two slices were prepared from every specimen and measured. It is reported that the spectra arise from domains approximately less than 1 µm deep (McCreery, 2000a, b). The epiderm is thicker than 1 µm in the mature wheat stem.

RESULTS AND DISCUSSION

Figure 1 shows three pairs of polarization NIR FT-Raman spectra of the stalk, sheath and nodes of the epidermal walls of wheat stem. A large difference between the two polarized spectra support the suggestion that there is an anisotropic arrangement of the biomacromolecules in the outer epidermal walls with respect to the longitudinal axis of the cell. The FT-Raman band assignments based on data from the literature are given in Table 1 (Marchessault and Sundararajan, 1983; Atalla and Agarwal, 1985; Agarwal and Ralph, 1997; Agarwal et al., 1999; Wilson et al., 2000).

Fig. 1.

Fourier transform-Raman spectra of wheat epidermis walls with parallel and perpendicular polarization: (A) stalk, (B) sheath, and (C) node. Raman intensity is given on a relative scale.

Table 1.

Fourier transform-Raman frequencies (cm⁻¹) of orientated macromolecules in the outer epidermal cell walls

Frequency (cm−1)	Contributor*	Band assignment
1630	L (=)	Ring conjugated C=C stretch of coniferaldehyde
1601	L (=)	Aryl ring stretching, symmetric
1456	C (+)	CH2 bending vibration
1377	C (+)	CH2 bending vibration
1335	C (+)	CH2 bending vibration
1267	L (=)	Guaiacyl ring breathing, Caryl–O stretching
1230	L (=)	Caryl–O stretching
1153	X (=)	The breathing vibration of the glucopyranose rings
1095	C (=)	Asymmetric COC stretching
1045	X (=)	C–C and C–O stretching

^*Orientation: (=), parallel; (+) perpendicular. C, cellulose; X, xylan; L, lignin.

For the cellulose, the Raman band at 1095 cm⁻¹ is assigned as the asymmetric COC stretching mode v_as (C–O–C), in which the vibration direction is aligned with the direction of the cellulose chains. The band is more intense with parallel polarization. The Raman band at 1120 cm⁻¹ was assigned to the skeletal vibrational modes v_s (C–O–C) of the glycosidic linkages of cellulose, xylan and lignin, which represent backbone vibrations of the polymer chain. The CH₂ bending vibration occurs at 1456, 1377 and 1335 cm⁻¹. In Fig. 1, their polarized directions of vibration are perpendicular to the backbone of the polymer chain, indicating more intense Raman signals with perpendicular polarization. This suggests that cellulose tends to be aligned parallel to the longitudinal axis of the cells in the epidermal cell walls of each wheat stem component.

Xylan is the major hemicellulose in the wheat stem cell wall. The main hemicellulosic chain consists of d-xylopyranose units linked by glycosidic β-(1→4)-linkages. The side chains are formed by l-arabinofuranosyl and d-xylopyranosyl units linked by α-(1→3)-linkages. The band at 1153 cm⁻¹ is the breathing vibration of the glucopyranose rings and the band at 1045 cm⁻¹ is associated with C–C and C–O stretching modes. The polarized direction of the modes' transition movement accords with the direction of the polymer chain. In all the figures, the bands are stronger in the transverse direction of the cells. In FTIR spectroscopy, xylan bands cannot be distinguished from cellulose bands (Aspinall, 1982). The Raman spectroscopy data provide good evidence that the xylan chains tend to lie perpendicular to the longitudinal axis of the cells in the epidermal components of wheat stem.

The band at 1600 cm⁻¹ in the Raman spectrum is due to the aromatic ring stretching modes, for which the polarized direction of transition movement accords with vibrations of modes of the phenylpropane units of lignin. The intensity of this band is lower when the direction of polarization is parallel to the longitudinal axis of the cell, indicating that the aromatic rings are preferentially orientated in the transverse direction. The bands at 1267 and 1230 cm⁻¹ are typical of C_aryl–O bands in lignin. In lignin, the aromatic C–O stretching is localized in the methoxyl and phenol groups. In the guaiacyl unit of lignin, these bonds vibrate mainly in the direction of the axis of the phenylpropane unit. Their intensity is greater in the transverse direction (Fig. 1A, C), indicating that the phenylpropane units are orientated in this direction in the epidermal cell walls of wheat stalks and nodes. These interpretations are supported by the observation that the 1630-cm⁻¹ band, which represents the C=C stretching vibrations of the conjugated aromatic rings, follows a pattern similar to that of the 1600-cm⁻¹ band in Fig. 1A and C. These bands provide good evidence that the phenylpropane units of lignin in the epidermal cell walls are mainly arranged perpendicular to the longitudinal axis of the cells in wheat stalks and nodes. Figure 1B does not show any difference between the two polarized spectra from lignin. Therefore, there is no evidence of any preferential orientation of lignin in the epidermal cell walls of wheat sheath.

CONCLUSIONS

This investigation has shown that Raman scattering spectroscopy is an excellent method for the study of cell-wall architecture. Use of NIR-FT Raman spectroscopy has shown that there is a net orientation of cellulose, xylan and lignin in the walls of the epidermal cells in wheat stem. The net orientation of cellulose in the epidermal cell walls is parallel to the longitudinal axis of the cells. The xylan tends to arrange perpendicular to the longitudinal axis of the cells. The phenylpropane of lignin in the epidermal cell walls is mainly arranged perpendicular to the longitudinal axis of the cells in wheat stalk and node, but not in sheath. The implication of this is that cellulose, lignin and xylan form a network that contributes to the mechanical and structural properties of the cell wall. Such results are likely to have a major impact on the formulation of definitive models for the epidermal cell wall.