Analysis of wheat grain development using NIR spectroscopy

Journal of Cereal Science 56 (2012) 31e38

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Analysis of wheat grain development using NIR spectroscopy András Salgó*, Szilveszter Gergely Department of Applied Biotechnology and Food Science, Budapest University of Technology and Economics, Szent Gellért tér 4, H-1111 Budapest, Hungary

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 February 2012 Received in revised form 24 April 2012 Accepted 25 April 2012

The scope of the present investigation was to detect maturation processes in wheat seed nondestructively with special respect to changes in moisture, carbohydrate and protein content and natural hydration/dehydration processes. During seed development, many biochemical, enzymatic and morphological changes occur under highly hydrated conditions. The ratio of different water species (high density water (HDW) with weaker hydrogen bonding and low density water (LDW) with stronger hydrogen bonding) changed drastically during maturation and their transitions could be followed with high sensitivity in specific regions of NIR spectra. In the maturing seed, two combination bands (Water I and Water III) were strong indicators of changes in water content while a first overtone (Water II) band gave a weaker response to change in moisture content. Three carbohydrate absorption bands showed different dynamics of carbohydrate (starch, fructan) accumulation and breakdown. Carbohydrate I represents starch accumulation during maturation based on the vibrations of intermolecular hydrogen-bonded OeH groups in polysaccharides. Carbohydrate II peak represents the of OeH stretching and CeC stretching vibrations in water-soluble carbohydrates while Carbohydrate III peak describes the changes in CeH stretching and deformation bands of poly-and mono-/oligosaccharides. Two protein absorption bands were identified (at 2055e2065 nm identified as amide A/II and at 2175 e2180 nm identified as amide I/III). These showed characteristic changes related to the accumulation of proteins and formation of the gluten network. The Amide A/II peak represents protein network formation during maturation based on the vibrations of inter-chain hydrogen-bonded NeH groups in polypeptides. The Amide I/III absorption band describes protein accumulation and the interactions of gliadins and glutenins that form the gluten network. NIR spectroscopy is shown to be effective in monitoring plant physiological processes both qualitatively and quantitatively, while the spectra also contain hidden information that can be used to define the stage of development of the wheat seed. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Maturation Seed development NIR spectroscopy Physiological process

1. Introduction Seed development, or maturation, is a phase of the life cycle of the plant and is often called the ripening process. Complex morphological, structural, metabolic and enzymatic processes take place in the developing seed in a very high moisture environment. Changes in the solubility of different hydrocolloids are influenced by the formation and polymerisation of dry materials such as starch and by the interactions between oligosaccharides and polymers of hydrocolloids and water. Water is essential for the whole maturation process, not only as an active agent, but also as a solvent providing a semiliquid medium for metabolic (enzymic) processes (Simon, 1984).

* Corresponding author. Tel.: þ36 1 4633854; fax: þ36 1 4633855. E-mail address: [email protected] (A. Salgó). 0733-5210/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2012.04.011

The migration of water in plant tissues is hindered by cell membranes, cell organelles and by interactions with different solutes and macromolecules. The most comprehensive description of both bulk and perturbed water models has been reviewed by Robinson et al. (1996). NIR spectroscopic methods (Iwamoto and Kawano, 1992; Norris, 1992) have been developed to measure the status and the changes of water in biological samples and food systems. These methods are sensitive enough to detect the changes of water not only as free solvent but also in highly associated macromolecular systems (Cassells et al., 2007; Evans et al., 1999; Wesley and Blakeney, 2001). NIR spectroscopy provides insights into the hydration and dehydration process of physiological events (Lovász et al., 1994; Salgó et al., 1995) in biological samples, and provides an understanding of the dynamic biochemical and enzymatic changes that are associated with water. During the different stages of maturation, the amount and composition of carbohydrates change in wheat seed due to the

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accumulation or depletion of different mono-, oligo- and polysaccharides (Kumar and Singh, 1981). Over the last three decades plant physiologists have developed models which describe the changes that occur in different sugars in wheat seeds during maturation. The amounts of water-soluble carbohydrates (monosaccharides, sucrose and fructans) increase greatly immediately after the onset of anthesis, showing a pronounced maximum at about 12/14 days after anthesis (DAF); thereafter, a progressive decrease occurs until 26/28 DAF after which the sugar pool is maintained at a relatively constant level, which is necessary for continued starch and cell wall synthesis (Cerning and Guilbot, 1973; Chanda et al., 1999; Escalada and Moss, 1976; Housley and Daughtry, 1987; Hurkman and Wood, 2011; Kumar and Singh, 1981; Paradiso et al., 2007; Toole et al., 2010). NIR spectroscopy is widely used to follow the chemical, physical, technological or physiological processes that affect the structure and composition of carbohydrates (Cassells et al., 2007). The quality of grain proteins (mainly storage proteins) affects the viscoelastic properties that enable dough to be processed into different foodstuffs (e.g., bread, biscuit, pasta, or noodles). The time course of synthesis and accumulation of proteins (and other components) strongly influence the functional and end-use quality of wheat (Shewry et al., 2009). A model has been developed to describe the changes that occur in different proteins during maturation of the grain. The proportions of water-soluble albumins and salt-soluble globulins (nearly 200 proteins, Hurkman et al., 2009) decline during grain filling while the amounts of gliadins and glutenins (gluten storage proteins) increase and they become aggregated into polymers with different sizes and solubilities (Carceller and Aussenac, 1999; Daniel and Triboï, 2002). The extractable polymeric proteins (EPP) include smaller aggregates and monomeric proteins, mainly gliadins and salt-water-soluble protein. By contrast, the non-extractable polymeric proteins (UPP) consist mostly of large aggregates of glutenins and gliadins. The EPP fraction shows a linear accumulation until the beginning of the dehydration phase, while the UPP fraction shows a slow linear increase up to this point. In the early phase of grain desiccation, the solubilities of proteins change significantly: the quantity of EPP decreases while the quantity of the UPP increases rapidly (Carceller and Aussenac, 1999, 2001; Daniel and Triboï, 2002). The hyperaggregation model therefore recognises at least two distinct levels of aggregation in the grain (Hamer and van Vliet, 2000; Rhazi et al., 2003). The first (molecular) level relates to the formation of EPP as covalent polymers are connected via disulphide bridges by a slow mechanism that operates until grain desiccation. At a second (mesoscopic) level larger aggregates (i.e., UPP) are formed by entanglement, stabilised by hydrogen bonding and additional disulphide bridges, which leads to the formation of a gluten network. According to this hypothesis, grain desiccation promotes the formation of the molecular level aggregates by facilitating in particular the formation of inter-chain hydrogen bonding between the repetitive regions of glutenin subunits. The fine details of NIR spectra can be used to predict the stage of growth and provide information on the biochemical events (Gergely and Salgó, 2007). The aim of the present paper is to summarise the most relevant data, knowledge and conclusions derived from NIR spectroscopy of the maturing wheat seed, drawing on our previously reported studies (Gergely and Salgó, 2003, 2005, 2007; Salgó et al., 1995). 2. Experimental Six winter wheat varieties with different harvest dates (GK Öthalom, Bánkúti 1201, Jubilejnaja 50, Mv 23, Fatima, and Mv 15) were grown in field trials at the Agricultural Research Institute of the Hungarian Academy of Sciences (ARIHAS), Martonvásár, during

1997. Primary ear samples were collected two or three times weekly, with 16 sampling dates covering the entire maturation period of 41 days (12e53 DAF). Because of the high biological variability of the samples, six independent ear samples were collected at each time. The seeds were collected from ears directly after sampling and analysed in intact form. The samples obtained on the first sampling date (12 DAF) differed from the others in that the immature kernels were so small that they had to be prepared together with their bracts (palea and lemma); at later stages 40e60 seeds were taken from each ear and the average fresh weights of the samples (mg seed1) were measured. Five independent scans were recorded from each sample using an NIRSystems Model 6500 monochromator system (Foss-NIRSystems, Silver Spring, MD, USA) NIR spectrometer, fitted with a sample transport module and standard sample cups. Samples were scanned (32 scans co-added) from 1100 to 2498 nm in reflectance mode (R mode: PbS detector). Data were collected every 2 nm (700 data points per spectrum) and the raw spectra were transformed into second derivatives (D2OD) using a 10 nm segment and 0 nm gap size. The second order finite-difference derivative requires two values to be specified: the length of the segment and the length of the gap between segments. The second derivative calculation begins by identifying three segments at one end of the spectrum, each separated from the other by a gap. Average absorbance values are calculated for the first, second, and third segments (A, B and C, respectively). The second derivative value computed as A  2B þ C is assigned to the midpoint of the second segment. The whole sequence of three segments and two gaps is then shifted by one data point and the calculations repeated until a second derivative value has been calculated for all data points in the spectrum. A segment size of 10 nm was adopted because preliminary results using segment sizes of 4, 10, and 20 nm showed no significant alterations to water peaks in second derivative spectra. Second derivative spectra have a negative peak that exactly matches the maximum (positive peak) of an absorbance band in log 1/R and these negative peaks were used to monitor moisture effects in the wheat seeds. The moisture content of intact, fresh seeds was determined in triplicate using oven drying (105  C for 4 h) immediately after collecting the spectra to avoid moisture loss. The nitrogen content of whole, dried seeds was measured in triplicate by combustion using a LECO FP-528 ProteineNitrogen Analyser (LECO Corp., St Joseph, MI, USA). Because the composition of wheat is characterised by a high content of starch, a significant protein content, and low and fairly constant contents of lipid and ash (Lásztity, 1999) (82%, 14%, 2%, 2% on dry weight basis, respectively) (Triboï and TriboïBlondel, 2002), the measured contents of moisture and protein allowed an estimate of the carbohydrate concentration as % dry mattere% protein (Hruschka, 1992). The remaining fresh samples and the dried materials were then frozen (15  C). To identify the most characteristic (i.e., most highly resolved) carbohydrate peaks during maturation, the major constituents of the grain [unmodified wheat starch (Sigma Chemical Co., St Louis, MO, USA), wheat gluten and gliadin (ICN Biomedicals, Irvine, CA, USA)] were also scanned as pure fractions using the same settings as for the wheat kernels. In addition, the NIR spectra of distilled water and aqueous solutions of some mono- and disaccharides [fructose, glucose and sucrose (Reanal Finechemical Co., Budapest, Hungary)] were collected in transmittance mode (T mode: PbS detector) in a 1 mm cuvette at room temperature, using dilution series of 0, 40, 100, 200 and 400 g L1. Spectral and reference data were processed using NIRSystems Spectral Analysis Software (NSAS), Ver. 3.30.

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3. Results and discussion The changes in the moisture content, the net dry matter and the calculated contents of starch and nitrogen in the maturing wheat seeds are shown in Fig. 1 (according to Gergely and Salgó, 2007). The data are expressed as % of fresh weight and concentration is determined by NIR spectroscopy through BeereLambert law (Osborne and Fearn, 1986). The moisture content, expressed as percent of fresh weight (Fig. 1a), decreased in a linear fashion throughout the period of measurement in accordance with published data (Morris et al., 1991). The plot of dry weight content as percent fresh weight is shown in Fig. 1b. The concentration of dry matter is affected both by the absolute amount of the dry material (as dissolved material), and also by the quantity of water (as solvent). The observed changes allowed us to define three phases of development over the period from 15 to 53 DAF. In the first phase (15e23 DAF), the percent dry weight gradually increases, because the rate of dry matter accumulation is higher than the rate of water accumulation. In the second phase (23e38 DAF), the increase in dry matter accumulation continues with the amount of moisture remaining relatively constant. In the final phase (38e53 DAF), the absolute dry weight per seed remains constant but the percent dry weight increases due to the loss of water by desiccation. The curves shown in Fig. 1c relate to calculated values for carbohydrate concentration as discussed in the Experimental section. The apparently linear increase in carbohydrate concentration masks the fact that both synthesis and degradation of starch and other carbohydrates occur. In accordance with previous observations, the nitrogen content of wheat grain during the cell division phase is derived largely from non-protein nitrogen and non-storage proteins, while the amounts of protein calculated from nitrogen analysis during the grain filling period mainly reflect storage protein accumulation (Huebner et al., 1990; Triboï et al., 2003). During the first and the second stages of grain development (between 15 and 38 DAF until the end of the cell expansion phase), the nitrogen content expressed as mg seed1

70 60 50 40 30 20 10 0 12 15 18 21 24 27 30 33 36 39 42 45 48 51

Three characteristic water absorption bands were studied in detail (Gergely and Salgó, 2003). These were the wavelength regions (a) between 1890 and 1920 nm (Water I: the combination of the OeH stretching band and the OeH bending band, n1,3 þ n2), (b) from 1400 to 1420 nm (Water II: the first overtone of the OeH stretching band, 2n1,3), and (c) from 1150 to 1165 nm (Water III: the combination of the first overtone of the OeH stretching band and the OeH bending band, 2n1,3 þ n2). The cut off wavelengths for the regions that encompass the three water bands were specified based on the local minimum values of D2OD spectra. Fig. 3 shows second derivative values for the Water I, II and III absorption bands for the wheat variety Bánkúti 1201. The absolute amount of water is proportional to the magnitude of the negative peak, while the wavelength shift indicates changes of associations of water. The Water I peak changed dramatically during maturation as shown in Fig. 3a according to previous observations (Gergely and Salgó, 2003). In the early stage of maturation (between 12 and 26 DAF), the water peak showed a shift towards the lower wavelength range (1890e1900 nm) as well as low D2OD values indicating a higher concentration of “free” water in the seed. The level of “free” water decreased in the 26e33 DAF period with little (approximately 2 nm) or no shift in wavelength. A characteristic shift in this water peak to higher wavelength values (15e20 nm), as

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3.1. Changes in water

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(data not shown) is linearly proportional to the amount of total protein accumulated (Fig. 1d) (Gergely and Salgó, 2007). Although changes in amino acid composition are known to occur during seed development, the protein content of the grain was calculated from the total nitrogen values by multiplying by a conversion factor of 5.7 (Stenram et al., 1990; Triboï et al., 2003). Changes in water, protein and carbohydrate content (Fig. 2a) and their ratios (Fig. 2b) during maturation are shown in Fig. 2. Specific regions in the NIR spectra allow the changes in concentrations of water, carbohydrates and proteins to be determined.

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Fig. 1. Changes in composition for seed samples of different six wheat varieties during maturation (% of fresh weight). (a) moisture content, (b) average dry weight, (c) carbohydrates, (d) nitrogen content - ¼ GK Öthalom, : ¼ Bánkúti 1201, C ¼ Jubilejnaja 50, , ¼ Mv 23, O ¼ Fatima, B ¼ Mv 15 (Taken from Gergely and Salgó, 2007 with permission).

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days after flowering Fig. 2. Changes in moisture, protein and carbohydrate content for seed samples (Jubilejnaja 50) during maturation expressed as (a) mg seed1 and (b) % of fresh ¼ water content, - ¼ protein content, F ¼ carbohydrate content (Taken weight. from Gergely and Salgó, 2007 with permission).

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well as a reduction in the D2OD values, was observed during the period between 33 and 53 DAF when the samples were desiccating. The local minimum values of this water peak showed characteristic patterns where not only in the amount but also in the strength of Hbonding water changed. The most sensitive changes in peaks were detected in the early phase of maturation (15e33 DAF) where rapid synthesis of carbohydrates, amino acids and oligopeptides and rapid hydration occur. In the later phase of maturation, the moisture content and the “free” character of water decreased steadily. It was suggested that the observed changes reflected the waterewater and waterematrix interactions during maturation (Gergely and Salgó, 2003). Changes during development can also be followed in another characteristic water band (Water II, between 1400 and 1420 nm) are also followed in Fig. 3b. The increase in the size of the negative peak in the D2OD showed that a significant increase in water content occurred in the early phase of maturation (15e23 DAF), followed by a rapid loss of water. This effect was observed in the spectrum between 1405 and 1410 nm, with a significant wavelength shift (from 10 to 15 nm) being observed only in the very late phase of maturation. The results indicated that changes in the amount and in type of water species were also reflected in the first overtone of the symmetric valence vibration (2n1,3), but that the extent and the sensitivity of changes were not as pronounced as in case of the Water I peak with the n2 term. The changes of the third important water peak (Water III) in the 1150e1165 nm region are summarised in Fig. 3c. During the early phase of maturation (15e33 DAF), only the concentration of water (i.e., the size of the water peak) changed, without any shift in the wavelength. In the later phase (33e53 DAF), a significant shift (approximately 10 nm) was detected indicating the effect of desiccation. Hydrogen bonding in water-based solutions can be influenced not only by temperature, but also by solutes (Ozaki and Wang, 1998; Segtnan et al., 2001). Chaotropic (structure breakers that

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Fig. 3. Changes of water peaks during maturation for variety Bánkúti 1201. (a) changes of water (Water I) peak in second derivative spectra of wheat seed in the 1890e1920 nm region, (b) changes of water peak (Water II) in second derivative spectra of wheat seed in the 1400e1420 nm region, (c) changes of water peak (Water III) in second derivative spectra of wheat seed in the 1150e1165 nm wavelength region (Taken from Gergely and Salgó, 2003 with permission).

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decrease the order of water) and kosmotropic (structure makers that increase order) materials produce two different states of solvation: water influenced by the solutes (hydration water) and undisturbed bulk water (called free water). Hydrogen bonding can be reduced by chaotropes or increased by kosmotropes (Galinski et al., 1997; Lever et al., 2001). Hydration water and bulk water are assumed to consist of two species: high density water (HDW: with weaker hydrogen bonding) and low density water (LDW: with stronger hydrogen bonding). The equilibrium between these components, can be shifted towards HDW by charged and polar (hydrophilic) groups or toward LDW by nonpolar (hydrophobic) groups (Chalikian, 2001). During the first phase (15e33 DAF), the wheat seed is a waterbased dilute solution of chaotropic monomers (i.e., charged and polar mono- and/or oligosaccharides and amino acids) and the equilibrium can be shifted towards HDW by hydrophilic monomers. During the second phase (33e53 DAF), starch and storage proteins accumulate rapidly (Lásztity, 1996). These biopolymers are less hydrophilic and more kosmotropic compared to the monomers used for their synthesis and the water peaks are therefore shifted to higher wavelengths (i.e., hydrogen bonds become stronger). 3.2. Changes in carbohydrates Changes in carbohydrates were detected in three wavelength regions (a) between 1585 and 1595 nm (labelled Carbohydrate I), (b) from 2270 to 2280 nm (Carbohydrate II), and (c) from 2325 to 2335 nm (Carbohydrate III). Fig. 4a summarises the changes in the intensity of the negative peak for Carbohydrate band I according to Gergely and Salgó (2005). This peak allows starch damage to be detected as it reflects the intermolecular hydrogen-bonded hydroxyl groups in

starch (Guy et al., 1996). Large A-type and small B-type starch granules are formed in wheat endosperm from approximately 4 and 14 DAF respectively, and then both types of granules grow until maturity (Peng et al., 2000). In the early phase, the high water environment in the wheat seed supports the weak association of starch molecules within the granules, which then become more closely associated as the water content decreases. The waterbinding capacity of the starch decreases as the grain matures, demonstrating a closer association of the starch molecules in the granules, thus reducing the accessibility of free hydroxyl groups for hydrogen bonding to water and, simultaneously, an increase in the amount of intermolecular hydrogen-bonded hydroxyl groups (Kulp and Mattern, 1973). Changes in another characteristic carbohydrate band (Carbohydrate II, between 2270 and 2280 nm) during maturation are shown in Fig. 4b. The spectra for the Carbohydrate II band showed changes during the early phase of maturation (15e26 DAF), not only in the magnitude of the D2OD local minimum but also shift in wavelength. This shift was greatest between 12 DAF, where the seeds were so small that they had to be prepared together with their bracts (palea and lemma), and 15 DAF which was the earliest stage at which seeds could be isolated. The period from 16 to 26 DAF is characterised by a decrease in the content of watersoluble carbohydrates which is reflected in the inverse value of the local minimum of Carbohydrate II band (Fig. 4b). Water-soluble carbohydrates such as fructose, glucose and sucrose possess distinct absorption bands around 2275 nm (Chung and Arnold, 2000) due to combinations of OeH stretching and CeC stretching vibrations (Osborne and Fearn, 1986). This clear and consistent peak, which decreases gradually during maturation, was assumed to be associated with non-starch polysaccharides including soluble pentosans (Czuchajowska and Pomeranz, 1989). Sugars make

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Fig. 4. Changes of carbohydrate peaks of wheat seed during maturation. (a) Changes of inverse value of local minimum of Carbohydrate I peak of wheat seed for six varieties. - ¼ GK Öthalom, : ¼ Bánkúti 1201, C ¼ Jubilejnaja 50, , ¼ Mv 23, O ¼ Fatima, B ¼ Mv 15. (b) Changes of inverse value of local minimum of Carbohydrate II peak of wheat seed for six varieties. - ¼ GK Öthalom, : ¼ Bánkúti 1201, C ¼ Jubilejnaja 50, , ¼ Mv 23, O ¼ Fatima, B ¼ Mv 15. (c) Changes of inverse value of local minimum of Carbohydrate III peak of wheat seed for six varieties. - ¼ GK Öthalom, : ¼ Bánkúti 1201, C ¼ Jubilejnaja 50, , ¼ Mv 23, O ¼ Fatima, B ¼ Mv 15 (Taken from Gergely and Salgó, 2005 with permission).

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3.3. Changes in proteins The accumulation of proteins and their biochemical interactions in vivo are very complex throughout grain maturation, so it was decided to study only the higher structure of proteins through the common “backbone” vibrations rather than specific side chain vibrations according to Liu et al. (1995). The side chain vibrations cannot be precisely identified in a heterogeneous molecular system such as the developing wheat seed so the Protein I (2040e2160 nm) and Protein II (2160e2220 nm) peak regions which correspond to backbone vibrations were analysed in detail. Fig. 5a summarises the magnitude of the negative peak for amide A/II band during maturation (Gergely and Salgó, 2007). The combination band of the amide A/II varies most significantly with increasing concentration in the folded (native) state (Ozaki et al., 1999; Wang et al., 1998). Secondary bonds (mainly intramolecular hydrogen bonds) play an important role in maintaining the higher structure of native proteins and the loss of stabilising hydrogen bonds results in modifications to the native structure. An increase in intensity of the amide A/II peak occurred during the last phase of the maturation process when the formation of the protein network takes place due to inter-chain hydrogen bonds. A higher frequency shift in the amide A/II combination band to a significantly lower wavelength (caused by weakening or destruction of the hydrogen bonds in the amide groups) could be followed due to the sensitivity of amide A vibration to the strength of the hydrogen bonds general in proteins (Liu et al., 1994, 1995) and especially in wheat gluten (Cho et al., 1995). We observed a 10 nm shift of the amide A/II peak in the opposite direction (data not shown), showing that the system of hydrogen bonds became stronger during the formation of the protein network in maturing wheat, but some differences were also observed in the extent and timing of the band shift among the wheat varieties.

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inverse value of local minimum

a significant contribution to the dry weight of grains during the early stages of seed development (Paradiso et al., 2007), resulting in high osmotic pressure and an influx of water that swells the cells beyond their normal capacity (Dhaliwal and Sharma, 1986; Jenner et al., 1991). Increasing the synthesis of starch then results in a decrease in the sugar content (15e26 DAF). However, the presence of significant amounts of water-soluble carbohydrates in wheat seeds, even when starch accumulation has decreased (26e38 DAF), suggests that the supply of sugar precursors does not limit starch synthesis (Dhaliwal and Sharma, 1986; Jenner et al., 1991; Kumar and Singh, 1981). Changes in the third carbohydrate peak (Carbohydrate III, between 2325 and 2335 nm) are shown in Fig. 4c. This carbohydrate peak shares many of the characteristics observed for the Carbohydrate I and Carbohydrate II peaks. The Carbohydrate III band probably relates to the combination of the bond vibration of the CeH stretch and the CeH deformation (Apruzzese et al., 2000; Chung and Arnold, 2000; Law and Tkachuk, 1977; Osborne and Fearn, 1986). The Carbohydrate I peak can be unambiguously related to starch accumulation because it relates to vibrations of intermolecular hydrogen-bonded OeH groups in polysaccharides, which in wheat are starch and its constituents (amylose and amylopectin). By contrast, the combination of OeH stretching and CeC stretching vibrations that relate to the Carbohydrate II peak reflect dissolved water-soluble carbohydrates (monosaccharides, sucrose and fructans). It is probable that the relation of the Carbohydrate III peak to both sugars and polysaccharides can be explained by the presence of the CeH group in carbohydrates, which were presumably less susceptible to the interactions in the continuously changing environment present in the maturing wheat seed.

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days after flowering Fig. 5. Changes of amide peaks during maturation. (a) Changes of inverse value of local minimum of amide A/II peak of wheat seed for six varieties. - ¼ GK Öthalom, : ¼ Bánkúti 1201, C ¼ Jubilejnaja 50, , ¼ Mv 23, O ¼ Fatima, B ¼ Mv 15. (b) Changes of inverse value of local minimum of amide I/III peak of wheat seed for six varieties. - ¼ GK Öthalom, : ¼ Bánkúti 1201, C ¼ Jubilejnaja 50, , ¼ Mv 23, O¼ Fatima, B ¼ Mv 15 (Taken from Gergely and Salgó, 2007 with permission).

These changes in hydrogen bonding patterns have also been described using Fourier transform infrared (FT-IR) spectra of isolated gluten from developing wheat seeds as corresponding to fewer extended chains and more b-sheet structures caused by the dehydration process (Wright et al., 2000). Plant physiological and spectroscopic studies have confirmed that the trend shown by the amide A/II peak in Fig. 5a follows the changes of the UPP fraction. The quantity of the UPP fraction increased exponentially at the mesoscopic level of aggregation due to some reorganisation in the hydrogen bonding patterns caused by the loss of water influencing the structure of the gluten network. Fig. 5b summarises the magnitude of the negative peak for amide I/ III band during maturation for six wheat varieties. The amide I/III combination band shows an opposite effect to the amide A/II band, namely the intensity of the amide I/III band changes most significantly in the spectra of samples in the unfolded state. The amide I/III band is sensitive to the quantity of proteins that (partially) lack hydrogen bonds to stabilise their native structure. In the present case, the state of EPP (extractable polymer proteins) was similar during the accumulation period (between 15 and 38 DAF) with respect to the hydrogen bond network status compared to the final structure of gluten which has an extended system of hydrogen bonds. It was pointed out that the trend in the amide I/III peak in Fig. 5b describes the changes in EPP (covalent polymers connected by disulphide bonds), with linear accumulation of EPP during the period when aggregation is at a molecular level (until 38 DAF)

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followed by slower accumulation during grain desiccation (Gergely and Salgó, 2007). The amount of EPP started to decrease at the beginning of the period when larger aggregates appear (later than 39e42 DAF), due to accumulation of the molecular aggregates. Fig. 5b suggests that the concentration of EPP is constant throughout the desiccation, but it is necessary to bear in mind that decreases in EPP during this period would be masked by the decrease in moisture content.

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contributions. This work was supported by the projects titled “Health Promotion and Tradition: Development of raw materials, functional foods and technologies in cereal-based food chain” (Project ID: TECH-08-A3/2-2008-0425). This work is connected to the scientific program of the “Development of quality-oriented and harmonized RþDþI strategy and functional model at BME” project. This project is supported by the New Hungary Development Plan (Project ID: TÁMOP-4.2.1/B-09/1/KMR-2010-0002).

4. Conclusions Our results confirmed (Gergely and Salgó, 2003) that the combination of the Water I and III bands were more sensitive indicators of changes to water in the maturing wheat seed than the overtone Water II band and that all the three water absorption bands can be used to monitor the status and changes of water clusters during hydration/dehydration processes. The rapid NIR assessment of the content and status of water could help to evaluate the effects of stresses including waterlogging (Hossain et al., 2011). It was also confirmed that many absorption bands in NIR spectra that can be sensitive indicators of changes in different carbohydrates (e.g., starch and water-soluble carbohydrates) (Gergely and Salgó, 2005). Spectroscopic methods therefore offer the opportunity, and potentially the ability, to determine fine details of physiological processes. In addition to starch and water-soluble carbohydrates, the NIR spectra can also potentially be used to determine changes in the amounts and compositions of dietary fibre components such as b-glucans (Seefeldt et al., 2009) and arabinoxylans (Salgó et al., 2009). In fact, the spectra have many more hidden details that can help us to understand the biochemical background of processes in maturing wheat seed! The time course of changes in expression (Katagiri et al., 2011), synthesis, folding, trafficking (Tosi et al., 2009) and accumulation (Hurkman et al., 2009) of wheat proteins during maturation strongly affect the formation of the gluten network and different quality parameters. We have analysed two absorption bands of protein peptide bonds in detail, confirming that the amide A/II absorption band (around 2060 nm) followed the formation of the gluten network. It was pointed out that the development of the hydrogen bond network at the mesoscopic level of aggregation can be followed due to the sensitivity of the amide A band (NeH stretching) to the strength of the hydrogen bonds (Gergely and Salgó, 2007). It was also shown that no further accumulation of storage proteins occurred after 38 DAF, but that the EPP formed at the molecular level of aggregation was converted into UPP, building the network of gluten proteins which plays a significant role in the functional properties of wheat flour and dough. The amide I/III absorption band (around 2180 nm) gave full information about the molecular level of aggregation, which could be interpreted as a quantitative change with no EPP protein accumulation. Numerous studies have attempted to use NIR to study the effects of storage conditions (Cassells et al., 2007) or other treatments on protein structure using pure proteins as dry powders or in aqueous solution. Since the molecular environment and interactions in the mature wheat seedare complex, we could not draw extensive conclusions about the primary and secondary structures of proteins from the NIR spectra of whole grains directly, but we were able to obtain a view of the higher level changes in storage protein structures associated with the formation of the gluten network. Acknowledgements The authors gratefully acknowledge László Láng (ARIHAS) for organising the plant trials and Adi Flower for valuable

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