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Spatial Distribution of Protein and Phenolic Constituents in Wheat Grain as Probed by Confocal Raman Microspectroscopy
Journal of Cereal Science 32 (2000) 57–71 doi:10.1006/jcrs.2000.0314, available online http://www.idealibrary.com on
Spatial Distribution of Protein and Phenolic Constituents in Wheat Grain as Probed by Confocal Raman Microspectroscopy O. Piot∗, J.-C. Autran† and M. Manfait∗ ∗IFR 53, Laboratoire de Spectroscopie Biomole´culaire, UFR de Pharmacie, 1 rue du Mare´chal Juin, 51096 Reims cedex, France; †INRA, Unite´ de Technologie des Ce´re´ales et des Agropolyme`res, 34060 Montpellier, France Received 3 June 1999
ABSTRACT Confocal Raman microspectroscopy is an appropriate method to investigate the microscopic structure of a wheat grain. The technique does not require any destructive preparation of the sample and is rapidly performed. The high spatial resolution reveals molecular and chemical heterogeneity within cellular dimensions. Using specific vibration bands as markers, the technique permits reconstruction of spectral images. The different components of wheat (Triticum aestivum) were chemically and structurally characterised by Raman microspectroscopy. The work was focused on the protein content and composition of the starchy endosperm and on the composition of the aleurone cells walls in arabinoxylan and ferulic acid derivatives. Particular attention was given to these components because of their role in cohesion of the starch–protein matrix interface and of the endosperm–envelope interface. Confocal Raman microscopy was also used to follow the evolution of protein content and structure during grain development of various wheat varieties selected on the basis of hardness level and aptitude to separation of peripheral layers during milling. 2000 Academic Press
Keywords: Raman microspectroscopy, Triticum aestivum wheat, hardness, structure, phenolic acid, protein, starch.
INTRODUCTION Dry milling of wheat is the attempt to separate the anatomical parts of the grain as cleanly as possible and more importantly to recover maximum levels of starchy endosperm as flour with minimum contamination from outer layers (pericarp, aleurone layer) and germ. Milling quality therefore includes the ease with which wheat endosperm is fractured (fragmentation) into small particles composed of starch granules and protein bodies, and the cleanliness with which separation ∗ Corresponding author: Prof. Michel Manfait, Laboratoire de Spectroscopie Biomole´culaire, IFR 53, 1 rue du Mare´chal Juin, UFR de Pharmacie, 51096 Reims Cedex, France. Tel: 33 3 26 91 35 74; Fax: 33 3 26 91 35 50; E-mail: [email protected] 0733–5210/00/070057+15 $35.00/0
occurs between endosperm and peripheral tissues. In this perspective, milling quality more specifically depends on: the ratio of starchy endosperm to outer layers; endosperm hardness or friability; bran friability, bran thickness and regularity, and ease of separation between bran and endosperm. At the microscopic level, a basic difference is observed between the mode of fracture propagation between hard and soft type wheats: hard kernels tend to fracture at cell walls (or, when cells are broken, within the starch granules), whereas soft kernels fracture through the endosperm cells at the starch–protein interface, leaving the starch granules undamaged. Additionally, hard wheats are characterised by better bran clean-up (because fracture also follows cell walls between endosperm 2000 Academic Press
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and aleurone layer) and higher extraction rates for hard wheats, but hard wheat flours contain a significant amount of mechanically damaged starch granules. In contrast with the number of studies devoted to the second transformation of wheat (dough formation, baking quality), the physico–chemical bases of grain fractionation and fragmentation remain largely unexplained, despite of its considerable economical importance. Although an important effort was placed on development of methods for hardness assessment and on development of theories of hardness, molecular species involved at the grain interfaces have not been identified and the physico–chemical basis of the mode of propagation of the fracture during milling is still unclear. A better understanding of the molecular basis of the fracture zones should lead to an improvement of milling quality through either breeding of better varieties and selection of particular agricultural practices, or optimisation of milling processes, e.g. application of specific pretreatments during storage or at the grain tempering step. This issue represents an economic stake for the industrial milling companies and for the cereal producers, in view of either higher milling yields in conventional flours, or valorisation of other milling products with improved protein–starch separation. Our studies aimed at characterising the structure of the wheat kernel at the micrometer scale using Raman microspectroscopy. Protein content and composition of starchy endosperm and composition of aleurone cell walls were specifically investigated (Fig. 1). It is valuable to characterise aleurone cells walls because they are implicated in the separation of the envelope and the kernel. Optical spectroscopy has been successfully used in cereal science to characterise the components of cereal grains or straw. FTIR spectroscopy was employed to assess efficacy of chemical treatments applied to barley straw1. In the early 90s, the microstructure of wheat kernel sections was examined, by coupling a FTIR spectrometer and a microscope adapted to the infrared source2. This in situ measurement permitted the observation of differences in composition of the different botanical parts of a grain. Maps of these parts were constructed using protein or lipid IR marker bands. In 1998, a synchrotron IR source was used to improve the performance of in situ microspectroscopy of plant tissue3. Microspectro-
fluorimetry is also a very powerful spectroscopic technique to detect and to locate in situ specific components of the wheat kernel. The properties of autofluorescence of phenolic components were used to study the distribution of ferulic acid within aleurone cell walls4. Nevertheless, although the technique is very sensitive, it does not give structural information as Raman or FTIR spectroscopy permits. Raman microspectroscopy is not only a powerful technique to identify cereal components, but it also gives information about secondary structure and configuration of these proteins. For instance, the technique permits to determine the conformation of a non-specific wheat phospholipid transfer protein5, and to study the role of disulphide bridges in the stabilisation of the -helical structure. In addition, Raman microspectroscopy was used to determine the secondary structure and conformation of puroindolines, lipid-binding protein of wheat6. In the first part of this study, the efficiency of confocal Raman microscopy in characterising the components of the wheat kernel will be demonstrated. In order to report on the high spatial resolution of the technique, we will present a spectral image showing protein distribution within the starchy endosperm. We will look at the composition of the aleurone cell walls (where fractionation between starchy endosperm and peripheral tissues takes place). We will also characterise the components of the germ that is separated from flour and bran during the milling process, because it can be valorised on the basis of its nutritional properties. Using these preliminary results, we will attempt, in a second part, to correlate kernel hardness with secondary structure of proteins. MATERIALS AND METHODS Plant material Experiments were carried out on wheat (Triticum aestivum) samples supplied by INRA (Montpellier, France) and by Champagne Ce´re´ales (Reims, France). Mature wheat grains of 20 different varieties (from soft to hard types) were supplied by Champagnes Ce´re´ales in order to investigate differences in structure between soft and hard varieties. Wheat grains (from soft to hard types) were collected at different stages of maturation. Twenty
Micro-Raman study of wheat grain
59
Crease Kernel Pigment strand Envelope Embryonic axis Scutellum
Testa and hyaline band
Pericarp
}
Germ
Aleurone cells layer
Subaleurone endosperm
Central starchy endosperm
Endosperm cell walls
Aleurone cell walls
Figure 1
Starch granules in protein mix
Transverse section of wheat grain (Fleckinger, 1935)22.
varieties sown at two dates (before and after the winter) and grown under two different conditions (without and with anti-fungal treatment) were available. Kernels were collected in an experimental field of Champagne Ce´re´ales during the last phase of grain development (starting one month before the harvest). Because of the large variability of wheat grains, special care was given to sampling. Wheat grains were always collected at the same location in the field and at the same position on the ear (between one-third and one-half of the ear’s height). The collection of grains at different maturation dates was done in the same atmospheric conditions, with no
sampling under rainy or wet weather conditions. Grains were kept at controlled room temperature (20 °C) and hygrometry (30%). Various reference products such as pure ferulic acid (Sigma), arabinoxylan (purified according to Figueroa-Espinoza and Rouau7) (Fig. 2), protein fractions (extracted and purified according to Scheromm et al.8). Solid fractions of gliadin were used to record reference spectra of protein. Spectra of all amino acids were also recorded. These reference products give specific vibration bands that are useful to identify the composition of the protein in situ.
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Xylose
O
H
O
OH HO
O
H
O
H O
OH
H
O
H
OH
OH
OH OH
H
OH
OH
O
O H
H
O
OH
H
OH
O
H
n O
H
OH HO
H2C
H OH
Arabinose unit OH
O O
O
O
O
OH OH
OH
OH
OH OH OH
Ferulic acid derivative
O OCH3 Ester bond O
Figure 2 Arabinoxylans formed of xylose chains with arabinose ramifications. Ferulic acid molecules can be bound to arabinose units via an ester bond.
Sample preparation For Raman experiments carried out on kernels, spectra were recorded on 50 m thick-solid sections. Sectioning of kernels was carried out transversally in ice using a cryomicrotome. In this study, only transverse sections at the middle (half-height) of the kernels were considered, except in investigations on the germ. Mature kernels were softened by soaking in distilled water (4 h at ambient temperature) before cutting in order to avoid damaging the blade. Grains sampled during maturation were directly sectioned by cryomicrotome without prior tempering. Samples were examined, at controlled room temperature and hygrometry, immediately after sectioning. Spectroscopic technique Confocal Raman microscopy was conducted on kernel sections using a Labram dispersive mi-
crospectrometer (Dilor, France). The originality of the technique resides in the coupling between a Raman spectrometer and an optical microscope. The microspectrometer is equipped with a He/Ne laser delivering 8 mW of red light, an illumination optical unit, which permits a good illumination of the sample by the laser light, an optical microscope (High stability BX 40 Olympus, 100× (NA 0,9) objective is used), a collection optical unit, which takes into account the confocal hole, dispersive systems constituted of two gratings mounted on the same shaft, 1800 grooves/mm (holographic) and 600 grooves/mm (ruled or holographic), and a multichannel CCD detector of 1024×256 pixels. The spectral resolution depends on the slit size. In the typical conditions of our experiments, the resolution was 2 cm−1. The confocality is assured by a diaphragm located in the focal image plane of the sample, just before the input of the spectrograph. The ad-
Micro-Raman study of wheat grain
vantage of confocal sampling is a considerable reduction of the depth of focus and thus an increased discrimination in the z-direction. This means that with a confocal set-up one can separate the signal from each layer of a multi-layered sample, i.e. perform optical sectioning. For a wheat grain section, it can be useful to reduce the confocal hole to be sure of recoring a specific component. Moreover, although the depth of penetration in our wheat sample is difficult to evaluate, the reduction of the confocal pinhole permits, in all cases, to improve lateral spatial resolution. The system also comprises a computer-controlled XY stage that can be adapted to cover a limited zone. The acquisition software (Labspec) allows the recording of several spectra following the displacement of the sample. By using certain marker bands, spectral images can be generated on one or more particular components9–15. Spectral treatment In a Raman spectrum, each peak or band is characterised by its position, intensity and profile. The position is related to the frequency of a vibrational mode. The intensity is related to the number of scattering molecules and to the considered vibrational mode (stretching, deformation). The profile can be complex, as several vibrations can be so close that peaks are not distinct but form a larger band such as the amide I band. Spectral treatment is sometimes necessary to decompose the profile and to uncover vibrational information. After performing second derivative of the spectra to precisely locate the frequency of the vibrations, a curve-fitting algorithm allows us to calculate a theoretical spectrum that best fits the experimental one. The large band can then be considered as a sum of these Lorentzian and/ or these Gaussian functions. All the different mathematical operations (baseline correction, normalisation, subtraction, second derivative) are performed using the Labspec software. RESULTS Wheat grains present a high heterogeneity and an important variability. This variability was assessed by averaging Raman spectra recorded on several grains of the same sample. The statistical study of 20 spectra of the same grain component recorded at the same location (e.g. protein matrix of the
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central kernel, or aleurone–endosperm cell wall) showed that the variability did not exceed 5% in intensity of the same vibration (amide I band or ferulic doublet). Characterisation of the wheat grain microstructure Microstructure of the starchy endosperm or grain kernel Within the starchy endosperm cell, two principal types of Raman spectra are discernible (Fig. 3). The first spectrum corresponds to a starch granule [Fig. 3(a)]. It represents the vibrational signature of starch, with typical vibrations at 476 and 940 cm−1 corresponding to stretching vibration of the carbonated skeleton. This spectrum corresponds to the spectrum of pure starch. The second type of Raman spectrum corresponds to the proteins located at the interstices of the starch granule [Fig. 3(b) and 3(c)]. The typical Raman signature of protein, such as the amide I band around 1656 cm−1 and the phenylalanine ring vibration at 1003 cm−1 can be seen. Figure 3(a and b) corresponds respectively to the central and the subaleurone endosperm protein. The protein content is more important in the subaleurone region than in the central region of the endosperm. Figure 3(b and c) also presents a contribution of starch, indicated by a vibration at frequency 476 and 940 cm−1. By normalising on these vibrations and by subtracting the spectrum of the starch granule [Fig. 3(a)] from the spectrum of the protein content [Fig. 3(b or c)], we could obtain a difference spectrum (Fig. 4), corresponding to the protein alone, more easily resolved to characterise the primary and secondary protein structures. With regard to the primary structure, several amino acid residues are detected. Phenylalanine is represented by vibration frequencies at 620, 1003 (ring breathing) and 1030 cm−1. Tyrosine is represented by vibrational frequencies at 642 cm−1 and by the Fermi resonance doublet 828–853 cm−1. The peak–height intensity ratio of these two bands provides information about the environment of the tyrosine residue. The vibration at 853 cm−1 is more intense, suggesting that the tyrosine residues are mostly exposed and are likely to form intermolecular bonds with other components, such as starch or lipids. Tryptophan residues are represented by vibration frequencies
O. Piot et al.
8
476
62
Starch vibrations
940
Raman intensity (a.u.)
6
4
2
0
500
1500
1000 Raman shift (cm–1)
1656 Amide I
1447 CH2 bending 1504
1550 Trp
1205
1602 Glk
1265 1298 1340 Trp
Pentosan
828 Tyr 853 873 Trp (H bond sensitive) 923 C-C stretch
4
620 Phe 642 Tyr
6
723 Trans C-S stretch 757 Trp
S-S stretch
8
510 532
Raman intensity (a.u.)
10
1030 Phe 1076 1098
1003 Phe
12
Amide III
Figure 3 Constituents of the grain kernel: micro-Raman spectra of starch granule (spectrum a ––––), central starchy endosperm protein (spectrum b – – – –) and subaleurone starchy endosperm protein (spectrum c · · · · · ·).
2
0 500
1000 Raman shift (cm–1)
1500
Figure 4 Difference spectrum (spectrum 3c−spectrum 3a) corresponding to the protein content of the subaleurone starchy endosperm.
Micro-Raman study of wheat grain
63
Hydrogen bond
O C
O N H
Figure 5
CHR1
C
O N
CHR2
O
C
N
CHR3
C
O N
CHR4
C
N
H
H
H
H
γ-turn
β-turn (310 helix)
α-helix
π-helix
Hydrogen bond vs secondary conformation.
at 757, 873, 1340 and 1550 cm−1. The band at 873 cm−1 is sensitive to hydrogen bonding at the NH of the indole ring. The low frequency position is evidence of a strong hydrogen bond, which is the present case. The lack of a vibrational peak at 1362 cm−1 (a vibration forming a Fermi doublet with the 1340 cm−1 band) tends to indicate that, as with the tyrosine residues, the tryptophane domain is linked to other molecules than the protein. There is no intramolecular binding between protein domains. The intensity ratio of vibrations at 620 cm−1 (Phe) and 642 cm−1 (Tyr) gives an indication of the ratio of number of each amino acid residues in the protein. Here, it seems that there are as many phenylalanine as tyrosine residues. The secondary conformation of the protein can also be studied by means of Raman vibrations such as disulphide bridge stretching or the amide I band. Indeed, disulphide bridges give a stretching (s-s) vibration between 510 and 540 cm−1 depending of the conformational structure of the bond. In the difference spectrum, two bands are visible at 510 and 532 cm−1, respectively corresponding to a conformation of the bonds C-CS-S-C-C in gauche-gauche-gauche (510 cm−1) and trans-gauche-gauche (532 cm−1). But the most sensitive marker for the secondary structure of proteins is the amide I band (C=O stretching+NH in plane bending of the peptide bond). The decomposition of amide I band will be studied in more details in the next subsection (Study of grain ripening). This type of secondary conformation is directly correlated with the stability of the protein. Each type of structure corresponds to a specific type of hydrogen bond, as shown in Fig. 5. The significant Raman vibrations of the starchy
endosperm components are summarised in Table I. Spectral image The previous results show that components of wheat kernel have specific vibration bands. Using these marker bands, it is possible to regenerate a spectral image9–15. For instance, high spatial resolution mappings have been constructed to see protein distribution within the starchy endosperm. Figure 6 shows the conventional image of the starchy endosperm and the two spectral images corresponding respectively to marker bands at 476 cm−1 for starch and the amide I band of the protein. The spectral images have been computer generated from a 50×50 Raman spectral set taken at 2 m intervals. All the spectra have been baseline corrected, offset to zero. It can clearly be seen that the protein material is located around the starch granules. Moreover, spectral imaging shows that the central big granule visible on the conventional photograph is in fact composed of several smaller starch granules in contact with protein. Such an image also contains quantitative information. Aleurone cell walls Spectra of the aleurone cell walls are shown in Figure 7. The spectra correspond to the aleurone– aleurone wall (wall between two neighbouring aleurone cells), the aleurone–pericarp wall (wall between an aleurone cell and the outer tissue of the envelope) and the aleurone–starchy endosperm wall. Figure 7 also represents Raman spectra of reference products, pure ferulic acid and arabinoxylan. The pure ferulic acid Raman spectrum is principally characterised by a vibration at 1178 cm−1, and by the doublet (stretching
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Table I
Principal Raman vibration bands of the starchy endosperm 476 cm−1 940 cm−1
Stretching vibration of the carbonated skeletal
620, 1003 (ring breathing) 1030 cm−1 642 cm−1, the doublet 828 and 853 cm−1 757, 873, 1340 and 1550 cm−1 1650–1680 cm−1
Phenylalanine Tyrosine Tryptophane Amide I frequency depending on the conformation S-S Stretching frequency depending on the conformation
Starch Protein content
510 and 532 cm−1
Length Y (µm)
Conventional image
20 30 40
40
60 80 Length X (µm)
(b)
Length Y (µm)
20
40
20
40
100
50
50
20 10 0
1003
Intensity (a.u.)
941
Intensity (a.u.)
40
40 30
100 Length X (µm)
476
Length X (µm)
30
1656
Length Y (µm)
(a)
100
20 10 0
500
1000 1500 Wavenumber (cm–1)
500
1000 1500 Wavenumber (cm–1)
Figure 6 Conventional image of the central starchy endosperm and constructed spectral images from starch vibration (a) and from protein amide I band (b). The whiter the points, the more intense the Raman scattering is.
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1603
Micro-Raman study of wheat grain
1630 1433
1178
Pure ferulic acid
1273
8
1273 1311 1373 1432 1460
1088 1122 897
494
1595 1626
897
494 533
Arabinoxylans
1311 1373
1088 1122
2
1460
4
Ferulic doublet
1626
Normalisation on the arabinoxylanes vibrations (1088–1122 cm–1)
533
Raman intensity (a.u.)
1595
6
0 500
1000 Raman shift (cm–1)
1500
Figure 7 Constituents of the aleurone cell walls: micro-Raman spectra of aleurone–aleurone wall (– – –), of aleurone– endosperm wall (· · · · · ·) and of aleurone–pericarp wall (–––).
vibration C=C of the phenolic ring) at 1603 and 1630 cm−1. The arabinoxylan chains are characterised by Raman vibration frequencies at 495, 898, 1090–1123 cm−1 and other peaks at 1314, 1369 and 1464 cm−1. The arabinoxylan chains have been extracted from cell walls. They are composed of a xylose chain with arabinose ramifications where ferulic acid molecules are likely to be esterified (Fig. 2). The change in the environment of the ferulic acid molecule is indicated by a frequency shift of the ferulic doublet from 1603–1630 cm−1 in the pure form to 1595 and 1627 cm−1 in the esterified form. Spectra have been normalised using the arabinoxylan vibration band in the 1090–1123 cm−1 region. These spectra show presence of ferulic acid derivative and arabinoxylan chains in these walls. It was noticed that the ratio of arabinoxylan to
ferulic acid depends on the type of cell walls. The ferulic component is twice as important in the anticlinal aleurone–aleurone wall than in the periclinal walls, aleurone–endosperm or aleurone– pericarp wall. Raman vibrations of the aleurone cell walls components are summarised in Table II. Germ As shown in Figure 1, the germ is composed of two major parts, the embryonic axis (rudimentary roots and shoot) and the scutellum, which functions as a storage organ. Raman spectra were recorded on different domains of the germ, scutellum and embryonic axis. Three different types of spectra are discernible (Fig. 8). The first spectrum [Fig. 8(a)] corresponds to the scutellum, the second [Fig. 8(b)] to em-
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Principal Raman vibration bands of the aleurone cell walls components 495, 533, 898, 1090–1123, 1314, 1369, 1464 cm−1 1273, doublet 1596–1627 cm−1 1178, 1273, 1433 doublet 1603–1630 cm−1
1743 Lipid C=O stretch
1656 Amide I 1528
}
Ferulic content 1595 1626 1656
–1
1460
897
0
1373
1088 1122
1156
1003 Phe 1030 Phe
1438
Glx, Ser
Tyr
782 832 852
1
620 Phe 642 Tyr
2
520 S-S stretch
Raman intensity (a.u.)
3
723 Trans C-S stretch
4
1578 Cysteine 1609 His 1603 Glx 1630 Ser
Amide III
Pure ferulic acid
Arabinoxylans chains Ferulic acid derivative
1260 1301 1341 Trp
Walls of aleurone cell
1311
Table II
–2 500
1000 Raman shift (cm–1)
1500
Figure 8 Constituents of the germ: micro-Raman spectra of scutellum cell (a ––––) of the embryonic axis cell (b · · · · · ·) and of the germ–endosperm interface (c – · – · –).
bryonic axis and the third [Fig. 8(c)] to the wall at the interface between germ and the starchy endosperm. The lipid content of the germ is represented by the vibration at 1743 cm−1, corresponding to the stretching vibration of the carbonyl group. The protein content is very well expressed in these spectra. Concerning the primary structure, we again find characteristic vibrations of phenylalanine and tyrosine residues. The ratio of the vibration bands at 620 and 642 cm−1 is similar to the spectrum of kernel protein (Fig. 4). Spectra of the scutellum and of the embryonic axis differ in the spectral region 1550–1630 cm−1. A spectrum of the scutellum [Fig. 8(a)] presents two bands at 1603 and 1630 cm−1 assigned respectively to the glutamic acid and serine residues. A spectrum of
the embryonic axis [Fig. 8(b)] presents two bands at 1578 and 1609 cm−1 assigned respectively to the cysteine and histidine residues. The presence of cysteine is also confirmed by C-S stretching at 782 cm−1. Concerning the germ–endosperm interface [Fig. 8(c)], the Raman spectrum shows the presence of arabinoxylan (897, 1088–1122, 1311, 1373, and 1460 cm−1), and of ferulic acid derivative (ferulic doublet 1595 and 1626 cm−1). However, the ferulic residue content is much less important than in the aleurone cell walls. It is similar to the endosperm cell walls (arabinoxylan as reference product on Figure 7). Moreover, in this wall we notice the presence of a poor quantity of protein. Iiyama et al.16 proposed several types of covalent cross-links in plant cell wall. Certain structures are formed
Micro-Raman study of wheat grain
with covalent bonds between, on one hand, phenyl rings of the ferulic derivatives esterified to the polysaccharide and, on the other hand, phenyl rings of the tyrosine or phenylalanine residues.
Study of grain ripening Figure 9 shows Raman spectra of the protein content of the central kernel, as maturation proceeds, for two varieties of wheat of different hardnesses. The period studied, starting roughly one month before the harvest, corresponds to the period of ripening and dehydrating of the grain. It extends from the milky phase to the pasty phase of grain development. As for what is shown in Figure 3, the Raman spectra present, in addition to the Raman signature of proteins, vibrations of starch at 476 and 940 cm−1. All spectra have been normalised on these starch vibrations, and no signal from water vibrations is visible in the studied spectral region. Consequently, the measurements are independent of moisture. The first observation is that at the interstices of starch granules, the protein content is more important in the hard variety. It appears also that the maturation of all varieties wheat grains leads to an increase of interstitial protein of the kernel. In a more detailed analysis, we looked at the differences between Raman spectra recorded at two successive collecting dates. We compared the interstitial protein increase between two successive maturation steps, independently of the moisture content. For the soft variety, as a consequence of the interstitial protein, Raman spectra exhibit an increase of phenylalanine vibrations (620, 1003 and 1201 cm−1), of tyrosine vibrations (642, doublet 828–856 cm−1), of disulphide bridge stretching (510 and 525 cm−1), and of vibrations located at 866 and 1073 cm−1, that could correspond to glutamic acid residues. The amide I band increased slightly. For the hard variety, the protein increase was charactrised by similar changes on phenylalanine and tyrosine vibrations, and of disulphide bridge stretching. No change was detected for vibrations at 866 and 1073 cm−1 whereas the amide I band centred on 1656 cm−1 greatly increased. Moreover, the hard variety presented an important increase in arabinoxylan content (495, 898, 1090–1123, 1314, 1372, 1463 cm−1), in contrast to the soft variety. The arabinoxylan vibrations mask vibrations of amino acids such as of tryptophan (1334, 1361 cm−1). Concerning the
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increase of the tyrosine Fermi doublet, the vibration at 856 cm−1 increased much more than the one at 828 cm−1. This spectroscopic result suggests that, as maturation proceeds, the tyrosine residue of the protein tends to be more exposed and thus contributes to new intermolecular bonds with other molecular species such as lipids. Concerning the disulphide bridge, the stretching vibration at 510 cm−1 was found to be correlated with the C-S stretching at 660 cm−1, visible on the spectra, which is evidence of a gauche-gauchegauche conformation of the disulphide bridge. In Figure 10, we present the distribution of amide I band (stretching C=O+bending NH) in -helical structure (1656 cm−1) and in other configurations ( sheet 1669 cm−1, turn 1676 cm−1 and random coil 1665 cm−1). The decomposition of amide I band was performed by the curve fitting function of the Labspec software. The frequencies of the vibrations were localised by doing a second derivative of spectra. It appears that the -helical structure gets more important during the kernel ripening, and that the hard variety has a proportion in -helix much greater than the soft variety, at the same maturation step. The helical structure could be strengthened by the exposed position of the tyrosine residue, likely to be bonded to lipids. Moreover, the structural difference between proteins of a soft and hard endosperm could also be associated with a difference in the amino acid composition of the protein matrix, and more particularly in glutamic acid and phenylalanine contents. These represent the main difference of amino acids composition between durum wheat and soft wheat17. The values of the ratio (-helical structure:other structures) at different maturation stages are indicated in the Table III. DISCUSSION The discussion will include two sections dealing with biochemical and technological aspects, especially the correlation between kernel hardness and protein content or conformation, and technical aspects of confocal Raman microscopy and other spectroscopic techniques. Correlation between kernel hardness and protein content and structure Raman microscopy represents a new method of investigation of the texture of a wheat kernel and brings new information on the grain hardness.
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476
68
15
1656
1001
Raman intensity (a.u.)
940
20
Soft Triticum aestivum wheat 10
5 Hard Triticum aestivum wheat 0 500
1000 Raman shift (cm–1)
1500
Figure 9 Raman spectra of the central kernel protein recorded on grains of two types of hardness, at different maturation stages (––– 7 July, – – – 21 July, · · · · · · 27 July). Spectra are normalised on the starch vibrations at 476 and 940 cm−1.
Several theories of kernel hardness have been expressed. The protein matrix18 is likely to be involved into kernel hardness, the bonding or adherence between starch granules and protein matrix would imply the presence of specific proteins19,20 such as friabilin or puroindoline-b, although the interaction between protein and starch granule surface can also involve a lipid factor. We have shown by using confocal Raman microscopy that the kernel hardness is sensitive to the amount of proteins present at the interstices between starch granules as well as the protein conformation of these proteins. Raman spectra of the interstitial protein take into account not only the storage protein (gliadin and glutenin) but also the protein components (puroindoline-a and -b) adhering to the surface of the starch granule. The surface protein can indeed be detected by its tryptophan content6,21 at 757, 1340, 1361 cm−1 specific of the indole ring and at 1550 and 1580 cm−1 whose intensity depends on the hydrophobicity. Changes in endosperm hardness are associated to an increase in the content of -helical structure of kernel proteins, which corresponds to the most stable protein conformation. A more detailed ana-
lysis of difference spectra between two consecutive collecting dates permitted indeed to observe changes in the primary structure (amino acid residues composition) and to correlate these modifications with the stabilisation of the protein in -helix conformation. Perspectives Concerning the cohesion of the wheat grain, another component could intervene in the fractures of the kernel. As the starchy endosperm is divided into cells, several questions could be asked about the role of the endosperm cell walls in the fractures of the kernel. Raman microscopy could be efficient in characterising endosperm cell walls of soft and hard wheat, as well as walls of the aleurone cells layer. Other authors have reported that a lipid factor could be participating in the adhesion of protein matrix at the starch granule surface. Although the lipid components of the surface of starch granules have not yet been approached through Raman microspectroscopy in our study, it will be done in a future work. The possible implication of a lipid–protein complex will be studied by using the spectral region of 2900–3000 cm−1.
Micro-Raman study of wheat grain 70
Distribution of amide I band
Soft Triticum aestivum wheat 60 50 40 30 20 10 0
7 July
16 July
27 July, harvest
21 July
69
maturation of the wheat grains, aleurone cell walls strengthen by formation of ferulic dimers. The ferulic dimers consequently form bridges between two arabinoxylan chains. Raman spectra did not show such a phenomenon during the final period of kernel ripening (one month before the harvest). Differences between anticlinal and periclinal walls, as shown in Figure 7, are the only visible features on these spectra. Perhaps, investigations should be carried out at an earlier period of maturation, as the formation of the envelopes occurs earlier than that of the inside of the kernel. In addition, no significant differences were observed between soft, medium hard, or hard types of Triticum (data not shown).
Maturation stages 70
Performances of confocal Raman microspectroscopy compared with infrared and fluorescence techniques
Distribution of amide I band
Hard Triticum aestivum wheat 60 50 40 30 20 10 0
7 July
16 July
21 July
27 July, harvest
Maturation stages
Figure 10 Distribution of the central kernel protein structure in helical conformation (Φ) and in the other conformations ( sheet, turn or 310 helix and random coil) (C) at different maturation stages, for two wheat varieties of different hardness. Harvest on the 27 July.
Concerning the evolution of the aleurone cell layer during the studied maturation period, no significant modification of the aleurone cell layer was visible. Iiyama et al.16 reported that during the Table III
The high spatial resolution of confocal Raman microspectroscopy allows characterisation of the structure of a wheat grain at the m scale. Concerning the constituents of the starchy endosperm, Raman spectra of starch and of proteins are very discernible and, for instance, we confirmed the greater amount of protein in the subaleurone endosperm than in the central endosperm. Moreover, Raman microscopy is not only an analytical tool but also a technique that allows characterisation of the spatial structure of the grain components and the secondary conformation of proteins. Compared with FTIR2, confocal Raman microspectroscopy presents the assets of a better spatial resolution and an easier setting up. Indeed, it is not possible to couple a classic optical microscope with a FTIR spectrometer because of the absorption of the infra-red radiation by glass objectives. Moreover, in the infrared domain, the spatial resolution remains limited by the diffraction limit, at >10 m. Nevertheless, for a better understanding of structure and bonding of molecular species over a
Distribution of the secondary structure of the central kernel protein: values of the ratio [ helix]/[ sheet+ turn+random coil] and of the confidence intervals, at different maturation stages Maturation stages
Soft Triticum aestivum wheat Hard Triticum aestivum wheat
7 July 0·69 (0·62–0·77) 0·83 (0·75–0·9)
16 July 0·68 (0·62–0·76) 0·98 (0·89–1·08)
24 July 0·83 (0·75–0·9) 1·50 (1·36–1·67)
27 July 0·92 (0·83–1) 1·6 (1·5–1·8)
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10 m scale, it could be sometimes useful to use infrared spectroscopy as a complementary technique of Raman spectroscopy. Indeed, for molecular systems with a degree of symmetry, the vibrational bands that are weak in Raman will be strong in infrared and vice versa. In order to improve the spatial resolution of infrared spectroscopy, Wetzel et al.3 used a synchrotron radiation as a new kind of infra-red source. The brightness of the synchrotron source, the absence of thermal noise and the non-divergent characteristics allowed us to reach a spatial resolution comparable with that of Raman microspectroscopy. Nevertheless, such a setting up is difficult to realise and certainly much more expensive than Raman microspectrometer. Raman as well as infra-red spectroscopy detects radiation corresponding to transitions between vibration states of the molecule and consequently allows to obtain structural information of a molecule5,6. On the other hand, the fluorescence phenomenon spectroscopy involves transitions between electronic states of a molecule, so that fluorescence spectroscopy can be a selective analytical technique, but is limited in characterising structure or functional groups of the molecule. In a recent study of aleurone cell walls, Saadi et al.4 demonstrated the presence of a feruloylated molecular species in the aleurone cell wall by fluorescence microspectroscopy. A difference was then established in the fluorescence intensity depending on the wall localisation: the fluorescence was more intense in the anticlinal wall (aleurone– aleurone) than in the periclinal wall. Not only Raman microspectroscopy confirms this difference between anticlinal and periclinal, but also reveals the state of binding between ferulic acid and arabinoxylan. Further investigations with several models of xylose chains with ramifications of arabinose and ferulic ester are underway and the first results should allow to determine the structure of the cell walls (degree of arabinose ramification and number of ferulic esters in each type of cell walls). One of the most interesting features of Raman microscopy is its versatility to a particular study as it is possible to select the laser excitation which gives the better Raman scattering without parasitic fluorescence. For instance, in the seed coat and at the centre of the crease, an important fluorescence is induced by 632·8 nm excitation (He/Ne laser), which could correspond to pigments. For such
kind component, near infrared spectroscopy (800–1064 nm) could be more adequate. Depolarisation ratio measurements might also be useful to examine physical and chemical properties of certain components which would have an organised structure such as crystalline zone into cell walls. CONCLUSION We believe that confocal Raman microspectroscopy represents a real breakthrough in the study of the microstructure of cereal grains. The technique permits not only the detection of molecular species on the m-level scale, but also the acquisition of information on their structure and on their binding with neighbouring molecules. Furthermore, it allows characterisation of their distribution by reconstruction of spectral images which are more informative than optical microscopy. For instance, changes in secondary structure of interstitial proteins, in particular, an increase in the helical content, have been detected when the kernel hardens during grain ripening. Acknowledgements We wish to thank Champagne Ce´re´ales (Reims, France) and Unite´ de Technologie des Ce´re´ales et des Agropolyme`res, INRA (Montpellier, France) for the supply of wheat samples and purification of the reference products (arabinoxylan, ferulic acid, protein fractions) used for the calibration of Raman microspectroscopy.
REFERENCES 1. Stewart, D. and Morisson, I. M. FT-IR spectroscopy as a tool for the study of biological and chemical treatments of barley straw. Journal of the Science of Food and Agriculture 60 (1992) 431–436. 2. Wetzel, D. L. and Reffner, J. A. Using spatially resolved Fourier transform infrared microbeam spectroscopy to examine the microstructure of wheat kernels. Cereal Foods World 38 (1993) 9–20. 3. Wetzel, D. L., Eilbert, A., Pietrzak, L., Miller., S. and Sweat, J. Ultraspatially-resolved synchrotron infrared microspectroscopy of plant tissue in situ. Cellular and Molecular Biology 44 (1998) 145–167. 4. Saadi, A., Lempereur, I., Sharonov, S., Autran, J. C. and Manfait, M. Spatial distribution of phenolic materials in durum wheat (Triticum durum Desf.) grain as probed by confocal fluorescence spectral imaging. Journal of Cereal Science 28 (1998) 107–114. 5. De´sormeaux, A., Blochet, J. E., Pe´zolet, M. and Marion, D. Amino acid sequence of a non-specific wheat phospholipid transfer protein and its conformation as revealed
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by infrared and Raman spectroscopy. Role of disulphide bridges and phospholipids in the stabilisation of the helix structure. Biochimica et Biophysica Acta 1121 (1992) 137–152. Le Bihan, T., Blochet, J. E., De´sormeaux, A., Marion, D. and Pe´zolet, M. Determination of the secondary structure and conformation of puroindolines by infrared and Raman spectroscopy. Biochemistry 35 (1996) 12712– 12722. Figueroa-Espinoza, M.-C. and Rouau, X. Oxidative cross-linking of pentosans by a fungal laccase and horseradish peroxidase: Mechanism of linkage between feruloylated arabinoxylan. Cereal Chemistry 75 (1998) 259–265. Scheromm P., Martin G., Bergoin, A. and Autran, J.-C. Influence of nitrogen fertilization on the potential breadbaking quality of two cultivars differing in their responses to increasing nitrogen supplies. Cereal Chemistry 69 (1992) 664–670. Sharonov, S., Millot, J. M., Angiboust, J. F., Kokota, A., Da Silva, E. and Manfait, M. Microspectrofluorime´trie confocale a` balayage laser et imagerie spectrale sur cellules vivantes isole´es. In ‘Dynamique de la cellule vivante’ (X. Ronot, ed. ), Les Editions INSERM, Paris (1999) 99–108. Manfait, M. Me´thodes d’imagerie optique pour le metabolisme cellulaire. In ‘Pharmacocine´tique et Biodistribution du Me´dicament. The´rapie Ge´nique Antisens’ ( J. M. Lhoste et C. Helene, eds.), Les Editions INSERM, Paris (1997) pp 59–76. Sharonov, S., Chourpa, I., Valisa, P. and Manfait, M. Confocal Raman microscopic imaging of biological samples. In ‘Advances in Fluorescence Sensing Technology III’ (R. B. Thompson, ed.), Proceedings of SPIE— The International Society for Optical Engineering, Bellingham (1997) Vol. 2980, pp 210–216. Sockalingum, G. D., Chourpa, I., Millot, J. M., Nabiev, I., Sharonov, S. and Manfait, M. Optical microspectroscopy and imaging analysis: probing drugs and metal ions in living cells. In ‘Advances in spectroscopy’ (R. J. H. Clarck and R. E. Hester, eds), Biomedical applications of spectroscopy (1996) Chapter 2, pp 49–87. Feofanov, A., Sharonov, S., Valisa, P., Da Silva, E.,
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Nabiev, I. and Manfait, M. A new confocal stigmatic spectrometer for micro-Raman and micro-fluorescence spectral imaging analysis. Design and applications. Reviews of Scientific Instruments 66 (1995) 1–13. Sharonov, S., Nabiev, I., Chourpa, I., Feofanov, A., Valisa, P. and Manfait, M. Confocal three-dimensional scanning laser Raman/SERS/fluorescence microprobe. Spectral imaging and high-resolution applications. Journal of Raman Spectroscopy 25 (1994) 699–707. Barbillat, J., Da Silva, E., Delahaye, M., Dhamelincourt, P., Manfait, M. and Roussel, B. Recent development of confocal Raman mapping. In ‘XIII International Conference on Raman Spectroscopy’ (W. Kiefer, M. Cardona, G. Schaack, F. W. Schneider and H. W. Schrotter eds), John Wiley & Sons, Chichester (1992) pp 1074–1075. Iiyama, K., Bach-Tuyet Lam, T. and Stone, B. A. Covalent cross-links in the cell wall. Plant Physiology 104 (1984) 315–320. Wrigley, C. W. and Bietz, J. A. Protein and Amino Acids. In ‘Wheat: Chemistry and Technology’ (Y. Pomeranz, ed.), AACC, St Paul MN (1988) Vol. 1, pp 229–233. Stenvert, N. L. and Kingswood, K. The influence of the physical structure of the protein matrix on wheat hardness. Journal of the Science of Food and Agriculture 28 (1977) 11–19.´ Greenwell, P. and Schofield, J. D. A starch granule protein associated with endosperm softness in wheat. Cereal Chemistry 38 (1986) 379–380. Greenwell, P. and Brock, C. Identity of starch-granulesurface proteins (friabilins) of bread wheat with detergentsoluble lipid binding proteins from flour. Presented at the 78th Annual AACC Meeting, Miami, Florida, USA. Cereal Foods World 38 (1993) 615–616. Blochet, J. E., Chevalier, C., Forest, E. Pebay-Peroula, E., Gautier, M. F., Joudrier, P., Pe´zolet, M. and Marion, D. Complete amino acid sequence of puroindoline, a new basic protein with a unique tryptophan-rich domain, isolated from wheat endosperm by Triton X114 phase partitioning. FEBS Letters 329 (1993) 336–340. Fleckinger. Le grain de ble´. Bulletin de l’Ecole Franc¸aise de Meunerie 36 (1935) 30–35.
Spatial Distribution of Protein and Phenolic Constituents in Wheat Grain as Probed by Confocal Raman Microspectroscopy O. Piot∗, J.-C. Autran† and M. Manfait∗ ∗IFR 53, Laboratoire de Spectroscopie Biomole´culaire, UFR de Pharmacie, 1 rue du Mare´chal Juin, 51096 Reims cedex, France; †INRA, Unite´ de Technologie des Ce´re´ales et des Agropolyme`res, 34060 Montpellier, France Received 3 June 1999
ABSTRACT Confocal Raman microspectroscopy is an appropriate method to investigate the microscopic structure of a wheat grain. The technique does not require any destructive preparation of the sample and is rapidly performed. The high spatial resolution reveals molecular and chemical heterogeneity within cellular dimensions. Using specific vibration bands as markers, the technique permits reconstruction of spectral images. The different components of wheat (Triticum aestivum) were chemically and structurally characterised by Raman microspectroscopy. The work was focused on the protein content and composition of the starchy endosperm and on the composition of the aleurone cells walls in arabinoxylan and ferulic acid derivatives. Particular attention was given to these components because of their role in cohesion of the starch–protein matrix interface and of the endosperm–envelope interface. Confocal Raman microscopy was also used to follow the evolution of protein content and structure during grain development of various wheat varieties selected on the basis of hardness level and aptitude to separation of peripheral layers during milling. 2000 Academic Press
Keywords: Raman microspectroscopy, Triticum aestivum wheat, hardness, structure, phenolic acid, protein, starch.
INTRODUCTION Dry milling of wheat is the attempt to separate the anatomical parts of the grain as cleanly as possible and more importantly to recover maximum levels of starchy endosperm as flour with minimum contamination from outer layers (pericarp, aleurone layer) and germ. Milling quality therefore includes the ease with which wheat endosperm is fractured (fragmentation) into small particles composed of starch granules and protein bodies, and the cleanliness with which separation ∗ Corresponding author: Prof. Michel Manfait, Laboratoire de Spectroscopie Biomole´culaire, IFR 53, 1 rue du Mare´chal Juin, UFR de Pharmacie, 51096 Reims Cedex, France. Tel: 33 3 26 91 35 74; Fax: 33 3 26 91 35 50; E-mail: [email protected] 0733–5210/00/070057+15 $35.00/0
occurs between endosperm and peripheral tissues. In this perspective, milling quality more specifically depends on: the ratio of starchy endosperm to outer layers; endosperm hardness or friability; bran friability, bran thickness and regularity, and ease of separation between bran and endosperm. At the microscopic level, a basic difference is observed between the mode of fracture propagation between hard and soft type wheats: hard kernels tend to fracture at cell walls (or, when cells are broken, within the starch granules), whereas soft kernels fracture through the endosperm cells at the starch–protein interface, leaving the starch granules undamaged. Additionally, hard wheats are characterised by better bran clean-up (because fracture also follows cell walls between endosperm 2000 Academic Press
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and aleurone layer) and higher extraction rates for hard wheats, but hard wheat flours contain a significant amount of mechanically damaged starch granules. In contrast with the number of studies devoted to the second transformation of wheat (dough formation, baking quality), the physico–chemical bases of grain fractionation and fragmentation remain largely unexplained, despite of its considerable economical importance. Although an important effort was placed on development of methods for hardness assessment and on development of theories of hardness, molecular species involved at the grain interfaces have not been identified and the physico–chemical basis of the mode of propagation of the fracture during milling is still unclear. A better understanding of the molecular basis of the fracture zones should lead to an improvement of milling quality through either breeding of better varieties and selection of particular agricultural practices, or optimisation of milling processes, e.g. application of specific pretreatments during storage or at the grain tempering step. This issue represents an economic stake for the industrial milling companies and for the cereal producers, in view of either higher milling yields in conventional flours, or valorisation of other milling products with improved protein–starch separation. Our studies aimed at characterising the structure of the wheat kernel at the micrometer scale using Raman microspectroscopy. Protein content and composition of starchy endosperm and composition of aleurone cell walls were specifically investigated (Fig. 1). It is valuable to characterise aleurone cells walls because they are implicated in the separation of the envelope and the kernel. Optical spectroscopy has been successfully used in cereal science to characterise the components of cereal grains or straw. FTIR spectroscopy was employed to assess efficacy of chemical treatments applied to barley straw1. In the early 90s, the microstructure of wheat kernel sections was examined, by coupling a FTIR spectrometer and a microscope adapted to the infrared source2. This in situ measurement permitted the observation of differences in composition of the different botanical parts of a grain. Maps of these parts were constructed using protein or lipid IR marker bands. In 1998, a synchrotron IR source was used to improve the performance of in situ microspectroscopy of plant tissue3. Microspectro-
fluorimetry is also a very powerful spectroscopic technique to detect and to locate in situ specific components of the wheat kernel. The properties of autofluorescence of phenolic components were used to study the distribution of ferulic acid within aleurone cell walls4. Nevertheless, although the technique is very sensitive, it does not give structural information as Raman or FTIR spectroscopy permits. Raman microspectroscopy is not only a powerful technique to identify cereal components, but it also gives information about secondary structure and configuration of these proteins. For instance, the technique permits to determine the conformation of a non-specific wheat phospholipid transfer protein5, and to study the role of disulphide bridges in the stabilisation of the -helical structure. In addition, Raman microspectroscopy was used to determine the secondary structure and conformation of puroindolines, lipid-binding protein of wheat6. In the first part of this study, the efficiency of confocal Raman microscopy in characterising the components of the wheat kernel will be demonstrated. In order to report on the high spatial resolution of the technique, we will present a spectral image showing protein distribution within the starchy endosperm. We will look at the composition of the aleurone cell walls (where fractionation between starchy endosperm and peripheral tissues takes place). We will also characterise the components of the germ that is separated from flour and bran during the milling process, because it can be valorised on the basis of its nutritional properties. Using these preliminary results, we will attempt, in a second part, to correlate kernel hardness with secondary structure of proteins. MATERIALS AND METHODS Plant material Experiments were carried out on wheat (Triticum aestivum) samples supplied by INRA (Montpellier, France) and by Champagne Ce´re´ales (Reims, France). Mature wheat grains of 20 different varieties (from soft to hard types) were supplied by Champagnes Ce´re´ales in order to investigate differences in structure between soft and hard varieties. Wheat grains (from soft to hard types) were collected at different stages of maturation. Twenty
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Crease Kernel Pigment strand Envelope Embryonic axis Scutellum
Testa and hyaline band
Pericarp
}
Germ
Aleurone cells layer
Subaleurone endosperm
Central starchy endosperm
Endosperm cell walls
Aleurone cell walls
Figure 1
Starch granules in protein mix
Transverse section of wheat grain (Fleckinger, 1935)22.
varieties sown at two dates (before and after the winter) and grown under two different conditions (without and with anti-fungal treatment) were available. Kernels were collected in an experimental field of Champagne Ce´re´ales during the last phase of grain development (starting one month before the harvest). Because of the large variability of wheat grains, special care was given to sampling. Wheat grains were always collected at the same location in the field and at the same position on the ear (between one-third and one-half of the ear’s height). The collection of grains at different maturation dates was done in the same atmospheric conditions, with no
sampling under rainy or wet weather conditions. Grains were kept at controlled room temperature (20 °C) and hygrometry (30%). Various reference products such as pure ferulic acid (Sigma), arabinoxylan (purified according to Figueroa-Espinoza and Rouau7) (Fig. 2), protein fractions (extracted and purified according to Scheromm et al.8). Solid fractions of gliadin were used to record reference spectra of protein. Spectra of all amino acids were also recorded. These reference products give specific vibration bands that are useful to identify the composition of the protein in situ.
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Xylose
O
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O OCH3 Ester bond O
Figure 2 Arabinoxylans formed of xylose chains with arabinose ramifications. Ferulic acid molecules can be bound to arabinose units via an ester bond.
Sample preparation For Raman experiments carried out on kernels, spectra were recorded on 50 m thick-solid sections. Sectioning of kernels was carried out transversally in ice using a cryomicrotome. In this study, only transverse sections at the middle (half-height) of the kernels were considered, except in investigations on the germ. Mature kernels were softened by soaking in distilled water (4 h at ambient temperature) before cutting in order to avoid damaging the blade. Grains sampled during maturation were directly sectioned by cryomicrotome without prior tempering. Samples were examined, at controlled room temperature and hygrometry, immediately after sectioning. Spectroscopic technique Confocal Raman microscopy was conducted on kernel sections using a Labram dispersive mi-
crospectrometer (Dilor, France). The originality of the technique resides in the coupling between a Raman spectrometer and an optical microscope. The microspectrometer is equipped with a He/Ne laser delivering 8 mW of red light, an illumination optical unit, which permits a good illumination of the sample by the laser light, an optical microscope (High stability BX 40 Olympus, 100× (NA 0,9) objective is used), a collection optical unit, which takes into account the confocal hole, dispersive systems constituted of two gratings mounted on the same shaft, 1800 grooves/mm (holographic) and 600 grooves/mm (ruled or holographic), and a multichannel CCD detector of 1024×256 pixels. The spectral resolution depends on the slit size. In the typical conditions of our experiments, the resolution was 2 cm−1. The confocality is assured by a diaphragm located in the focal image plane of the sample, just before the input of the spectrograph. The ad-
Micro-Raman study of wheat grain
vantage of confocal sampling is a considerable reduction of the depth of focus and thus an increased discrimination in the z-direction. This means that with a confocal set-up one can separate the signal from each layer of a multi-layered sample, i.e. perform optical sectioning. For a wheat grain section, it can be useful to reduce the confocal hole to be sure of recoring a specific component. Moreover, although the depth of penetration in our wheat sample is difficult to evaluate, the reduction of the confocal pinhole permits, in all cases, to improve lateral spatial resolution. The system also comprises a computer-controlled XY stage that can be adapted to cover a limited zone. The acquisition software (Labspec) allows the recording of several spectra following the displacement of the sample. By using certain marker bands, spectral images can be generated on one or more particular components9–15. Spectral treatment In a Raman spectrum, each peak or band is characterised by its position, intensity and profile. The position is related to the frequency of a vibrational mode. The intensity is related to the number of scattering molecules and to the considered vibrational mode (stretching, deformation). The profile can be complex, as several vibrations can be so close that peaks are not distinct but form a larger band such as the amide I band. Spectral treatment is sometimes necessary to decompose the profile and to uncover vibrational information. After performing second derivative of the spectra to precisely locate the frequency of the vibrations, a curve-fitting algorithm allows us to calculate a theoretical spectrum that best fits the experimental one. The large band can then be considered as a sum of these Lorentzian and/ or these Gaussian functions. All the different mathematical operations (baseline correction, normalisation, subtraction, second derivative) are performed using the Labspec software. RESULTS Wheat grains present a high heterogeneity and an important variability. This variability was assessed by averaging Raman spectra recorded on several grains of the same sample. The statistical study of 20 spectra of the same grain component recorded at the same location (e.g. protein matrix of the
61
central kernel, or aleurone–endosperm cell wall) showed that the variability did not exceed 5% in intensity of the same vibration (amide I band or ferulic doublet). Characterisation of the wheat grain microstructure Microstructure of the starchy endosperm or grain kernel Within the starchy endosperm cell, two principal types of Raman spectra are discernible (Fig. 3). The first spectrum corresponds to a starch granule [Fig. 3(a)]. It represents the vibrational signature of starch, with typical vibrations at 476 and 940 cm−1 corresponding to stretching vibration of the carbonated skeleton. This spectrum corresponds to the spectrum of pure starch. The second type of Raman spectrum corresponds to the proteins located at the interstices of the starch granule [Fig. 3(b) and 3(c)]. The typical Raman signature of protein, such as the amide I band around 1656 cm−1 and the phenylalanine ring vibration at 1003 cm−1 can be seen. Figure 3(a and b) corresponds respectively to the central and the subaleurone endosperm protein. The protein content is more important in the subaleurone region than in the central region of the endosperm. Figure 3(b and c) also presents a contribution of starch, indicated by a vibration at frequency 476 and 940 cm−1. By normalising on these vibrations and by subtracting the spectrum of the starch granule [Fig. 3(a)] from the spectrum of the protein content [Fig. 3(b or c)], we could obtain a difference spectrum (Fig. 4), corresponding to the protein alone, more easily resolved to characterise the primary and secondary protein structures. With regard to the primary structure, several amino acid residues are detected. Phenylalanine is represented by vibration frequencies at 620, 1003 (ring breathing) and 1030 cm−1. Tyrosine is represented by vibrational frequencies at 642 cm−1 and by the Fermi resonance doublet 828–853 cm−1. The peak–height intensity ratio of these two bands provides information about the environment of the tyrosine residue. The vibration at 853 cm−1 is more intense, suggesting that the tyrosine residues are mostly exposed and are likely to form intermolecular bonds with other components, such as starch or lipids. Tryptophan residues are represented by vibration frequencies
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Figure 3 Constituents of the grain kernel: micro-Raman spectra of starch granule (spectrum a ––––), central starchy endosperm protein (spectrum b – – – –) and subaleurone starchy endosperm protein (spectrum c · · · · · ·).
2
0 500
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Figure 4 Difference spectrum (spectrum 3c−spectrum 3a) corresponding to the protein content of the subaleurone starchy endosperm.
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Hydrogen bond
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Figure 5
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at 757, 873, 1340 and 1550 cm−1. The band at 873 cm−1 is sensitive to hydrogen bonding at the NH of the indole ring. The low frequency position is evidence of a strong hydrogen bond, which is the present case. The lack of a vibrational peak at 1362 cm−1 (a vibration forming a Fermi doublet with the 1340 cm−1 band) tends to indicate that, as with the tyrosine residues, the tryptophane domain is linked to other molecules than the protein. There is no intramolecular binding between protein domains. The intensity ratio of vibrations at 620 cm−1 (Phe) and 642 cm−1 (Tyr) gives an indication of the ratio of number of each amino acid residues in the protein. Here, it seems that there are as many phenylalanine as tyrosine residues. The secondary conformation of the protein can also be studied by means of Raman vibrations such as disulphide bridge stretching or the amide I band. Indeed, disulphide bridges give a stretching (s-s) vibration between 510 and 540 cm−1 depending of the conformational structure of the bond. In the difference spectrum, two bands are visible at 510 and 532 cm−1, respectively corresponding to a conformation of the bonds C-CS-S-C-C in gauche-gauche-gauche (510 cm−1) and trans-gauche-gauche (532 cm−1). But the most sensitive marker for the secondary structure of proteins is the amide I band (C=O stretching+NH in plane bending of the peptide bond). The decomposition of amide I band will be studied in more details in the next subsection (Study of grain ripening). This type of secondary conformation is directly correlated with the stability of the protein. Each type of structure corresponds to a specific type of hydrogen bond, as shown in Fig. 5. The significant Raman vibrations of the starchy
endosperm components are summarised in Table I. Spectral image The previous results show that components of wheat kernel have specific vibration bands. Using these marker bands, it is possible to regenerate a spectral image9–15. For instance, high spatial resolution mappings have been constructed to see protein distribution within the starchy endosperm. Figure 6 shows the conventional image of the starchy endosperm and the two spectral images corresponding respectively to marker bands at 476 cm−1 for starch and the amide I band of the protein. The spectral images have been computer generated from a 50×50 Raman spectral set taken at 2 m intervals. All the spectra have been baseline corrected, offset to zero. It can clearly be seen that the protein material is located around the starch granules. Moreover, spectral imaging shows that the central big granule visible on the conventional photograph is in fact composed of several smaller starch granules in contact with protein. Such an image also contains quantitative information. Aleurone cell walls Spectra of the aleurone cell walls are shown in Figure 7. The spectra correspond to the aleurone– aleurone wall (wall between two neighbouring aleurone cells), the aleurone–pericarp wall (wall between an aleurone cell and the outer tissue of the envelope) and the aleurone–starchy endosperm wall. Figure 7 also represents Raman spectra of reference products, pure ferulic acid and arabinoxylan. The pure ferulic acid Raman spectrum is principally characterised by a vibration at 1178 cm−1, and by the doublet (stretching
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Table I
Principal Raman vibration bands of the starchy endosperm 476 cm−1 940 cm−1
Stretching vibration of the carbonated skeletal
620, 1003 (ring breathing) 1030 cm−1 642 cm−1, the doublet 828 and 853 cm−1 757, 873, 1340 and 1550 cm−1 1650–1680 cm−1
Phenylalanine Tyrosine Tryptophane Amide I frequency depending on the conformation S-S Stretching frequency depending on the conformation
Starch Protein content
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Figure 6 Conventional image of the central starchy endosperm and constructed spectral images from starch vibration (a) and from protein amide I band (b). The whiter the points, the more intense the Raman scattering is.
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1603
Micro-Raman study of wheat grain
1630 1433
1178
Pure ferulic acid
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8
1273 1311 1373 1432 1460
1088 1122 897
494
1595 1626
897
494 533
Arabinoxylans
1311 1373
1088 1122
2
1460
4
Ferulic doublet
1626
Normalisation on the arabinoxylanes vibrations (1088–1122 cm–1)
533
Raman intensity (a.u.)
1595
6
0 500
1000 Raman shift (cm–1)
1500
Figure 7 Constituents of the aleurone cell walls: micro-Raman spectra of aleurone–aleurone wall (– – –), of aleurone– endosperm wall (· · · · · ·) and of aleurone–pericarp wall (–––).
vibration C=C of the phenolic ring) at 1603 and 1630 cm−1. The arabinoxylan chains are characterised by Raman vibration frequencies at 495, 898, 1090–1123 cm−1 and other peaks at 1314, 1369 and 1464 cm−1. The arabinoxylan chains have been extracted from cell walls. They are composed of a xylose chain with arabinose ramifications where ferulic acid molecules are likely to be esterified (Fig. 2). The change in the environment of the ferulic acid molecule is indicated by a frequency shift of the ferulic doublet from 1603–1630 cm−1 in the pure form to 1595 and 1627 cm−1 in the esterified form. Spectra have been normalised using the arabinoxylan vibration band in the 1090–1123 cm−1 region. These spectra show presence of ferulic acid derivative and arabinoxylan chains in these walls. It was noticed that the ratio of arabinoxylan to
ferulic acid depends on the type of cell walls. The ferulic component is twice as important in the anticlinal aleurone–aleurone wall than in the periclinal walls, aleurone–endosperm or aleurone– pericarp wall. Raman vibrations of the aleurone cell walls components are summarised in Table II. Germ As shown in Figure 1, the germ is composed of two major parts, the embryonic axis (rudimentary roots and shoot) and the scutellum, which functions as a storage organ. Raman spectra were recorded on different domains of the germ, scutellum and embryonic axis. Three different types of spectra are discernible (Fig. 8). The first spectrum [Fig. 8(a)] corresponds to the scutellum, the second [Fig. 8(b)] to em-
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O. Piot et al.
Principal Raman vibration bands of the aleurone cell walls components 495, 533, 898, 1090–1123, 1314, 1369, 1464 cm−1 1273, doublet 1596–1627 cm−1 1178, 1273, 1433 doublet 1603–1630 cm−1
1743 Lipid C=O stretch
1656 Amide I 1528
}
Ferulic content 1595 1626 1656
–1
1460
897
0
1373
1088 1122
1156
1003 Phe 1030 Phe
1438
Glx, Ser
Tyr
782 832 852
1
620 Phe 642 Tyr
2
520 S-S stretch
Raman intensity (a.u.)
3
723 Trans C-S stretch
4
1578 Cysteine 1609 His 1603 Glx 1630 Ser
Amide III
Pure ferulic acid
Arabinoxylans chains Ferulic acid derivative
1260 1301 1341 Trp
Walls of aleurone cell
1311
Table II
–2 500
1000 Raman shift (cm–1)
1500
Figure 8 Constituents of the germ: micro-Raman spectra of scutellum cell (a ––––) of the embryonic axis cell (b · · · · · ·) and of the germ–endosperm interface (c – · – · –).
bryonic axis and the third [Fig. 8(c)] to the wall at the interface between germ and the starchy endosperm. The lipid content of the germ is represented by the vibration at 1743 cm−1, corresponding to the stretching vibration of the carbonyl group. The protein content is very well expressed in these spectra. Concerning the primary structure, we again find characteristic vibrations of phenylalanine and tyrosine residues. The ratio of the vibration bands at 620 and 642 cm−1 is similar to the spectrum of kernel protein (Fig. 4). Spectra of the scutellum and of the embryonic axis differ in the spectral region 1550–1630 cm−1. A spectrum of the scutellum [Fig. 8(a)] presents two bands at 1603 and 1630 cm−1 assigned respectively to the glutamic acid and serine residues. A spectrum of
the embryonic axis [Fig. 8(b)] presents two bands at 1578 and 1609 cm−1 assigned respectively to the cysteine and histidine residues. The presence of cysteine is also confirmed by C-S stretching at 782 cm−1. Concerning the germ–endosperm interface [Fig. 8(c)], the Raman spectrum shows the presence of arabinoxylan (897, 1088–1122, 1311, 1373, and 1460 cm−1), and of ferulic acid derivative (ferulic doublet 1595 and 1626 cm−1). However, the ferulic residue content is much less important than in the aleurone cell walls. It is similar to the endosperm cell walls (arabinoxylan as reference product on Figure 7). Moreover, in this wall we notice the presence of a poor quantity of protein. Iiyama et al.16 proposed several types of covalent cross-links in plant cell wall. Certain structures are formed
Micro-Raman study of wheat grain
with covalent bonds between, on one hand, phenyl rings of the ferulic derivatives esterified to the polysaccharide and, on the other hand, phenyl rings of the tyrosine or phenylalanine residues.
Study of grain ripening Figure 9 shows Raman spectra of the protein content of the central kernel, as maturation proceeds, for two varieties of wheat of different hardnesses. The period studied, starting roughly one month before the harvest, corresponds to the period of ripening and dehydrating of the grain. It extends from the milky phase to the pasty phase of grain development. As for what is shown in Figure 3, the Raman spectra present, in addition to the Raman signature of proteins, vibrations of starch at 476 and 940 cm−1. All spectra have been normalised on these starch vibrations, and no signal from water vibrations is visible in the studied spectral region. Consequently, the measurements are independent of moisture. The first observation is that at the interstices of starch granules, the protein content is more important in the hard variety. It appears also that the maturation of all varieties wheat grains leads to an increase of interstitial protein of the kernel. In a more detailed analysis, we looked at the differences between Raman spectra recorded at two successive collecting dates. We compared the interstitial protein increase between two successive maturation steps, independently of the moisture content. For the soft variety, as a consequence of the interstitial protein, Raman spectra exhibit an increase of phenylalanine vibrations (620, 1003 and 1201 cm−1), of tyrosine vibrations (642, doublet 828–856 cm−1), of disulphide bridge stretching (510 and 525 cm−1), and of vibrations located at 866 and 1073 cm−1, that could correspond to glutamic acid residues. The amide I band increased slightly. For the hard variety, the protein increase was charactrised by similar changes on phenylalanine and tyrosine vibrations, and of disulphide bridge stretching. No change was detected for vibrations at 866 and 1073 cm−1 whereas the amide I band centred on 1656 cm−1 greatly increased. Moreover, the hard variety presented an important increase in arabinoxylan content (495, 898, 1090–1123, 1314, 1372, 1463 cm−1), in contrast to the soft variety. The arabinoxylan vibrations mask vibrations of amino acids such as of tryptophan (1334, 1361 cm−1). Concerning the
67
increase of the tyrosine Fermi doublet, the vibration at 856 cm−1 increased much more than the one at 828 cm−1. This spectroscopic result suggests that, as maturation proceeds, the tyrosine residue of the protein tends to be more exposed and thus contributes to new intermolecular bonds with other molecular species such as lipids. Concerning the disulphide bridge, the stretching vibration at 510 cm−1 was found to be correlated with the C-S stretching at 660 cm−1, visible on the spectra, which is evidence of a gauche-gauchegauche conformation of the disulphide bridge. In Figure 10, we present the distribution of amide I band (stretching C=O+bending NH) in -helical structure (1656 cm−1) and in other configurations ( sheet 1669 cm−1, turn 1676 cm−1 and random coil 1665 cm−1). The decomposition of amide I band was performed by the curve fitting function of the Labspec software. The frequencies of the vibrations were localised by doing a second derivative of spectra. It appears that the -helical structure gets more important during the kernel ripening, and that the hard variety has a proportion in -helix much greater than the soft variety, at the same maturation step. The helical structure could be strengthened by the exposed position of the tyrosine residue, likely to be bonded to lipids. Moreover, the structural difference between proteins of a soft and hard endosperm could also be associated with a difference in the amino acid composition of the protein matrix, and more particularly in glutamic acid and phenylalanine contents. These represent the main difference of amino acids composition between durum wheat and soft wheat17. The values of the ratio (-helical structure:other structures) at different maturation stages are indicated in the Table III. DISCUSSION The discussion will include two sections dealing with biochemical and technological aspects, especially the correlation between kernel hardness and protein content or conformation, and technical aspects of confocal Raman microscopy and other spectroscopic techniques. Correlation between kernel hardness and protein content and structure Raman microscopy represents a new method of investigation of the texture of a wheat kernel and brings new information on the grain hardness.
O. Piot et al.
476
68
15
1656
1001
Raman intensity (a.u.)
940
20
Soft Triticum aestivum wheat 10
5 Hard Triticum aestivum wheat 0 500
1000 Raman shift (cm–1)
1500
Figure 9 Raman spectra of the central kernel protein recorded on grains of two types of hardness, at different maturation stages (––– 7 July, – – – 21 July, · · · · · · 27 July). Spectra are normalised on the starch vibrations at 476 and 940 cm−1.
Several theories of kernel hardness have been expressed. The protein matrix18 is likely to be involved into kernel hardness, the bonding or adherence between starch granules and protein matrix would imply the presence of specific proteins19,20 such as friabilin or puroindoline-b, although the interaction between protein and starch granule surface can also involve a lipid factor. We have shown by using confocal Raman microscopy that the kernel hardness is sensitive to the amount of proteins present at the interstices between starch granules as well as the protein conformation of these proteins. Raman spectra of the interstitial protein take into account not only the storage protein (gliadin and glutenin) but also the protein components (puroindoline-a and -b) adhering to the surface of the starch granule. The surface protein can indeed be detected by its tryptophan content6,21 at 757, 1340, 1361 cm−1 specific of the indole ring and at 1550 and 1580 cm−1 whose intensity depends on the hydrophobicity. Changes in endosperm hardness are associated to an increase in the content of -helical structure of kernel proteins, which corresponds to the most stable protein conformation. A more detailed ana-
lysis of difference spectra between two consecutive collecting dates permitted indeed to observe changes in the primary structure (amino acid residues composition) and to correlate these modifications with the stabilisation of the protein in -helix conformation. Perspectives Concerning the cohesion of the wheat grain, another component could intervene in the fractures of the kernel. As the starchy endosperm is divided into cells, several questions could be asked about the role of the endosperm cell walls in the fractures of the kernel. Raman microscopy could be efficient in characterising endosperm cell walls of soft and hard wheat, as well as walls of the aleurone cells layer. Other authors have reported that a lipid factor could be participating in the adhesion of protein matrix at the starch granule surface. Although the lipid components of the surface of starch granules have not yet been approached through Raman microspectroscopy in our study, it will be done in a future work. The possible implication of a lipid–protein complex will be studied by using the spectral region of 2900–3000 cm−1.
Micro-Raman study of wheat grain 70
Distribution of amide I band
Soft Triticum aestivum wheat 60 50 40 30 20 10 0
7 July
16 July
27 July, harvest
21 July
69
maturation of the wheat grains, aleurone cell walls strengthen by formation of ferulic dimers. The ferulic dimers consequently form bridges between two arabinoxylan chains. Raman spectra did not show such a phenomenon during the final period of kernel ripening (one month before the harvest). Differences between anticlinal and periclinal walls, as shown in Figure 7, are the only visible features on these spectra. Perhaps, investigations should be carried out at an earlier period of maturation, as the formation of the envelopes occurs earlier than that of the inside of the kernel. In addition, no significant differences were observed between soft, medium hard, or hard types of Triticum (data not shown).
Maturation stages 70
Performances of confocal Raman microspectroscopy compared with infrared and fluorescence techniques
Distribution of amide I band
Hard Triticum aestivum wheat 60 50 40 30 20 10 0
7 July
16 July
21 July
27 July, harvest
Maturation stages
Figure 10 Distribution of the central kernel protein structure in helical conformation (Φ) and in the other conformations ( sheet, turn or 310 helix and random coil) (C) at different maturation stages, for two wheat varieties of different hardness. Harvest on the 27 July.
Concerning the evolution of the aleurone cell layer during the studied maturation period, no significant modification of the aleurone cell layer was visible. Iiyama et al.16 reported that during the Table III
The high spatial resolution of confocal Raman microspectroscopy allows characterisation of the structure of a wheat grain at the m scale. Concerning the constituents of the starchy endosperm, Raman spectra of starch and of proteins are very discernible and, for instance, we confirmed the greater amount of protein in the subaleurone endosperm than in the central endosperm. Moreover, Raman microscopy is not only an analytical tool but also a technique that allows characterisation of the spatial structure of the grain components and the secondary conformation of proteins. Compared with FTIR2, confocal Raman microspectroscopy presents the assets of a better spatial resolution and an easier setting up. Indeed, it is not possible to couple a classic optical microscope with a FTIR spectrometer because of the absorption of the infra-red radiation by glass objectives. Moreover, in the infrared domain, the spatial resolution remains limited by the diffraction limit, at >10 m. Nevertheless, for a better understanding of structure and bonding of molecular species over a
Distribution of the secondary structure of the central kernel protein: values of the ratio [ helix]/[ sheet+ turn+random coil] and of the confidence intervals, at different maturation stages Maturation stages
Soft Triticum aestivum wheat Hard Triticum aestivum wheat
7 July 0·69 (0·62–0·77) 0·83 (0·75–0·9)
16 July 0·68 (0·62–0·76) 0·98 (0·89–1·08)
24 July 0·83 (0·75–0·9) 1·50 (1·36–1·67)
27 July 0·92 (0·83–1) 1·6 (1·5–1·8)
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O. Piot et al.
10 m scale, it could be sometimes useful to use infrared spectroscopy as a complementary technique of Raman spectroscopy. Indeed, for molecular systems with a degree of symmetry, the vibrational bands that are weak in Raman will be strong in infrared and vice versa. In order to improve the spatial resolution of infrared spectroscopy, Wetzel et al.3 used a synchrotron radiation as a new kind of infra-red source. The brightness of the synchrotron source, the absence of thermal noise and the non-divergent characteristics allowed us to reach a spatial resolution comparable with that of Raman microspectroscopy. Nevertheless, such a setting up is difficult to realise and certainly much more expensive than Raman microspectrometer. Raman as well as infra-red spectroscopy detects radiation corresponding to transitions between vibration states of the molecule and consequently allows to obtain structural information of a molecule5,6. On the other hand, the fluorescence phenomenon spectroscopy involves transitions between electronic states of a molecule, so that fluorescence spectroscopy can be a selective analytical technique, but is limited in characterising structure or functional groups of the molecule. In a recent study of aleurone cell walls, Saadi et al.4 demonstrated the presence of a feruloylated molecular species in the aleurone cell wall by fluorescence microspectroscopy. A difference was then established in the fluorescence intensity depending on the wall localisation: the fluorescence was more intense in the anticlinal wall (aleurone– aleurone) than in the periclinal wall. Not only Raman microspectroscopy confirms this difference between anticlinal and periclinal, but also reveals the state of binding between ferulic acid and arabinoxylan. Further investigations with several models of xylose chains with ramifications of arabinose and ferulic ester are underway and the first results should allow to determine the structure of the cell walls (degree of arabinose ramification and number of ferulic esters in each type of cell walls). One of the most interesting features of Raman microscopy is its versatility to a particular study as it is possible to select the laser excitation which gives the better Raman scattering without parasitic fluorescence. For instance, in the seed coat and at the centre of the crease, an important fluorescence is induced by 632·8 nm excitation (He/Ne laser), which could correspond to pigments. For such
kind component, near infrared spectroscopy (800–1064 nm) could be more adequate. Depolarisation ratio measurements might also be useful to examine physical and chemical properties of certain components which would have an organised structure such as crystalline zone into cell walls. CONCLUSION We believe that confocal Raman microspectroscopy represents a real breakthrough in the study of the microstructure of cereal grains. The technique permits not only the detection of molecular species on the m-level scale, but also the acquisition of information on their structure and on their binding with neighbouring molecules. Furthermore, it allows characterisation of their distribution by reconstruction of spectral images which are more informative than optical microscopy. For instance, changes in secondary structure of interstitial proteins, in particular, an increase in the helical content, have been detected when the kernel hardens during grain ripening. Acknowledgements We wish to thank Champagne Ce´re´ales (Reims, France) and Unite´ de Technologie des Ce´re´ales et des Agropolyme`res, INRA (Montpellier, France) for the supply of wheat samples and purification of the reference products (arabinoxylan, ferulic acid, protein fractions) used for the calibration of Raman microspectroscopy.
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