Article pubs.acs.org/JAFC

Heterogeneous Structure and Spatial Distribution in Endosperm of High-Amylose Rice Starch Granules with Different Morphologies Canhui Cai,†,‡ Jun Huang,†,‡ Lingxiao Zhao,†,‡ Qiaoquan Liu,†,‡ Changquan Zhang,†,‡ and Cunxu Wei*,†,‡ †

Key Laboratories of Crop Genetics and Physiology of the Jiangsu Province and Plant Functional Genomics of the Ministry of Education, Yangzhou University, Yangzhou 225009, China ‡ Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China ABSTRACT: Starch granules from high-amylose cereal mutants or transgenic lines usually have different morphologies. It is not clear whether the structure and spatial distribution of starch granules with different morphologies in endosperm is homogeneous or heterogeneous. In the present study, the structure and spatial distribution in endosperm of morphologically different starch granules from high-amylose transgenic rice line (TRS) were investigated. The TRS endosperm had individual, aggregate, elongated, and interior hollow starch granules. The individual and interior hollow granules had the lowest and the highest amylose content and gelatinization resistance, respectively, among the four types of granules. The individual granules were mainly distributed in the middle of the endosperm; the aggregate granules in the starchy endosperm cells between the subaleurone layer and the middle of the endosperm; the elongated granules in the peripheral starchy endosperm cells adjacent to the subaleurone layer; and the interior hollow granules in the subaleurone layer cells. KEYWORDS: high-amylose rice, heterogeneous starch granule, morphology, structure, spatial distribution in endosperm

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INTRODUCTION Starch consists of two main components: linear amylose and the highly branched amylopectin. Amylose content has a pronounced effect on the physicochemical properties and applications of starch. For nutritional purposes, starch is classified into three types: rapidly digestible starch, slowly digestible starch, and resistant starch (RS).1 RS is a portion of starch that cannot be hydrolyzed in the upper gastrointestinal tract and functions as a substrate for bacterial fermentation in the large intestine.1 In general, RS content of granular starch is positively correlated with the level of amylose.2 RS has been reported to provide many health benefits for humans, because RS-enriched food can lower the glycemic and insulin responses and reduce the risk of developing type II diabetes mellitus, obesity, and cardiovascular disease.3 Previous studies suggest that the endosperm of high-amylose cereal is usually rich in RS and has potential health benefits through high amylose content.4−6 Therefore, there is a growing interest in highamylose cereal crops with many varieties developed using mutation or transgenic breeding approaches.5−9 High-amylose cereal starches always show a markedly different morphology and physicochemical properties compared with waxy and normal starches.5,9−12 For example, normal maize starches are angular or spherical in shape, while the high-amylose maize ae and GEMS-0067 mutants contain about 7% and 32% elongated starch granules, respectively.10 Normal wheat and barley seeds have lenticular large starch granules and spherical small ones, but wheat and barley seeds with a high-amylose content, caused by the inhibition of starch branching enzymes, are found to form some sickle-shaped starch granules with a hollow interior.5,9,11 Isolated normal rice starch consists entirely of individual starch granules, whereas high-amylose rice mutant Goami 2 (previously known as © 2014 American Chemical Society

Suweon 464) starch consists of two populations: large voluminous bodies consisting of tightly packed small subgranules and individual granules.12 Starches from high-amylose cereal endosperm usually consist of morphologically different granules.5,9−12 However, the structure and spatial distribution in the endosperm of morphologically different starch granules have seldom been reported in previous research. It is not clear whether the structure and spatial distribution in the endosperm of morphologically different starch granules are homogeneous or heterogeneous. Recently, the study of ae, a high-amylose mutant maize, showed that heterogeneity in chemical and physical structure can be observed within the same granule, between granules within cells, and spatially within the kernel.13,14 The morphologically different starch granules from high-amylose maize (HYLON VII) also show significant differences in structure and gelatinization properties.15 A high-amylose transgenic rice line (TRS) has been developed by antisense RNA inhibition of both starch branching enzyme I (SBE I) and SBE IIb in our laboratory, which yields a starch with an amylose content of about 60%.6,16 TRS kernels are rich in RS and have shown significant potential to improve the health of the large bowel in rats.6 Results from our microstructure and ultrastructure studies have revealed that TRS starch consists of morphologically different granules.17 However, the structure of these granules, especially their spatial distribution in the endosperm, has not been reported. Received: Revised: Accepted: Published: 10143

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by using a vortex mixer. The suspension was transferred onto a slide, covered with a coverslip, and sealed with nail polish to prevent moisture loss during heating. The sealed specimen was then mounted on a Kitazato hot stage apparatus and observed under a long focus M Plan Semi Apochromat objective (50× magnification) using an Olympus polarized microscope during heating. The hot stage was heated from 25 to 50 °C at a heating rate of 5 °C/min and from 50 to 100 °C at a heating rate of 1 °C/min. The gelatinization temperatures were measured according to the method of Konik-Rose et al.20 with some modifications. The starch slurry was photographed under polarized light using an Olympus DP72 CCD camera during heating from 50 to 100 °C at 1 °C intervals. Three group experiments were carried out for every type of starch granule. For every group experiment, more than 100 granules of every type of heterogeneous starch granules were analyzed from 10 to 30 photographs. For every granule, the temperature at which the granule lost birefringence was recorded. The granule number was counted at the lost birefringence temperature. Plotting the granule number percentage of birefringence loss against the temperature produced a gelatinization curve. The initial, middle, and end gelatinization temperatures were obtained from the gelatinization curve when 5%, 50%, and 95% of granules had lost their birefringence. The swelling of starch granule during heating was viewed under normal light and photographed using an Olympus DP72 CCD camera from 40 to 95 °C at 5 °C intervals. The area of starch granule was analyzed from the micrograph with a JEDA 801D morphological image analysis system (Jiangsu JEDA Science-Technology Development Co., Ltd., Nanjing, China). Fifty starch granules were measured for every type of heterogeneous starch granule. The starch granule swelling was presented as area swelling percentage (ASP) from ungelatinized granule at 40 °C and calculated by the equation ASP (%) = At/Ai × 100, where Ai and At represented the area of starch granule at initial (40 °C) and specific testing temperature, respectively. Kernel Sections for Light Microscopy. In order to soften the kernels, mature kernels were first fixed in a fixation solution (2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.2) for 2 h at room temperature and then held overnight at 4 °C. The softened kernels were transversely cut with a razor blade at the midregion of the kernel to obtain thin tissue blocks. The tissue blocks were then fixed in the same fixation solution again for 48 h at 4 °C. After fixation, the blocks were washed 3 times with phosphate buffer, successively dehydrated in gradient ethanol, and embedded in LR White Resin. The semithin sections of 2 μm thickness were cut with a glass knife on a Leica Ultrathin Microtome (EM UC7), stained with iodine solution (25% glycerol, 0.07% I2, 0.14% KI) for 10 min in darkness, and observed with an Olympus BX53 light microscope equipped with a CCD camera. Statistical Analysis. One-way analysis of variance (ANOVA) and Tukey’s test (p < 0.05) were used for statistical analysis of the difference of properties of TQ starch granule, TRS starch individual granule, TRS starch aggregate granule, TRS starch elongated granule, and TRS starch interior hollow granule using the SPSS 16.0 Statistical Software Program. All the replications were technical replicates.

In the present study, we aim to use various microscopy techniques to characterize the structure of morphologically different starch granules isolated from TRS mature kernels. The mature kernels will be mapped in situ using light microscopy to identify the spatial distribution of morphologically different granules in the endosperm. This study could increase our understanding of heterogeneous starch granules from highamylose cereal and would provide important information on the formation of heterogeneous starch granules resulting from kernel development.

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MATERIALS AND METHODS

Plant Materials. An indica rice cultivar Te-qing (TQ) and its transgenic line (TRS) with high amylose and RS contents were used in this study. TRS was generated from TQ after transgenic inhibition of both SBE I and SBE IIb through an antisense RNA technique and was homozygous for the transgene.6 TQ and TRS were cultivated in the transgenic close experiment field of Yangzhou University, Yangzhou, China. Starch Isolation. Starches were isolated from TQ and TRS mature kernels according to the method of Wei et al.16 with some modifications. Brown rice seeds were steeped in distilled water at 4 °C overnight and homogenized with ice-cold water in a home blender. The homogenate was squeezed through five layers of cotton cloth. The residue was homogenized and squeezed twice more in a mortar with a pestle to facilitate the release of starch granules. The combined extract was filtered with 100-, 200-, and 400-mesh sieves and centrifuged at 3000g for 10 min. The yellow gel-like layer on top of the packed white starch granule pellet was carefully scraped off and discarded. The process of centrifugation separation was repeated several times until no dirty material existed. The precipitated starch was further washed with anhydrous ethanol two times, dried at 40 °C, ground into powders, and passed through a 100-mesh sieve. Light Microscopy. A starch suspension (1%, w/v) was prepared with 50% glycerol. 10 μL of starch suspension was placed on the microscope slide and covered with a coverslip. The starch granule shape and Maltese cross were viewed with an Olympus BX53 polarized light microscope equipped with a CCD camera under normal and polarized light. Iodine-stained starches were prepared and observed according to the method of Evans et al.18 with some modifications. 5 mg of starch was stained in 0.5 mL of iodine solution (0.5% NaAc buffer, pH4.5, 25% glycerol, 0.04% I2, 0.06% KI) for 30 min in darkness. 10 μL of iodine-stained starch suspension was first viewed under normal light, and then the same field was viewed under polarized light. All the iodine-stained starch granules were photographed under the same conditions in order to compare the polarization colors. Iodine-stained starches had four kinds of polarization colors: blue, fuchsia, red, and interior dark blue and exterior brown. For one type of heterogeneous starch granules, 50 granules were randomly chosen to be analyzed for polarization color for one experiment. The experiments were performed in triplicate. Confocal Laser Scanning Microscopy. Starch granules were stained with the fluorophore APTS (8-amino-1,3,6-pyrenetrisulfonic acid) and prepared for confocal laser scanning microscope (CLSM) essentially as previously described by Blennow et al.19 Images were recorded on a CLSM (LSM 710, Carl Zeiss MicroImaging GmbH) using a 488 nm laser line for excitation, and light was detected in the interval from 500 to 535 nm. For morphology observation, laser power capacity and master gain were adjusted to maximum saturation. To compare the fluorescence intensity, the laser power was kept constant at 1%, and images were recorded at the master gain of 620 for all starch granules. The conditions allowed all granules to have no saturation of the detector for every dot in all granules. For every type of heterogeneous starch granules, 60 granules were randomly chosen to be recorded. Image analysis was performed using the Carl Zeiss ZEN 2010 software. Hot Stage Microscopy. Starch suspension was prepared by suspending about 10 mg of starch in 1.0 mL of double distilled water

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RESULTS AND DISCUSSION Morphology of Heterogeneous Starch Granules. Starch granules isolated from TQ and TRS mature kernels were observed under normal and polarized light (Figure 1). Most TQ starch granules were regular and polygonal (Figure 1A,C) with all of them showing one central hilum and one typical Maltese cross (Figure 1a,c). Therefore, TQ starch granules were morphologically homogeneous. TQ starch is a compound starch that separates during isolation into many subgranules.17 In the present study, the TQ starch granules isolated were the subgranules of compound starch that appeared as individual granules. As found previously,17 TRS starch granules showed significantly different morphologies (Figure 1B,b). These 10144

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elongated granule exhibited no birefringence (Figure 1f4). This phenomenon has also been reported elsewhere in high-amylose maize starch granules.15,21 The percentage of elongated starch granules increases with the increasing amylose content in highamylose maize.10 The TRS interior hollow granules were mostly irregular and deformed, and exhibited extremely weak to no birefringence (Figure 1g), which was the same as interior hollow starch granules from high-amylose SGP-1 null wheat.22 Aggregate, elongated, and interior hollow starch granules have been observed in high-amylose rice,12,17 maize,10,15 wheat,5,9 and barley.11 However, the mode of formation of interior hollow granules is not clear at present. Birefringence patterns are the result of orderly aligned polymers or crystallites. Different birefringence patterns show various radial orientations and alignments of the crystallites’ regions within the granules.23,24 In the present study, the TQ and TRS individual starch granules and the subgranules of TRS aggregate starch granule had a typical Maltese cross in the hilum, indicating that the starch molecules were aligned in a radial order centered at the hilum and perpendicular to the granule surface.21 In high-amylose maize, about 36% and 55% of elongated granules have partial birefringence and no birefringence, respectively.15 In high-amylose wheat and barley, over 90% of interior hollow starch granules are not birefringent.5,11 In the present study, the TRS elongated and interior hollow starch granules had weak or no birefringence, indicating that the starch molecules of granules were not radially aligned, which might result from the high amylose concentrations in these granules. The amylose molecules of granules are not expected to be specifically uniformly oriented.19 The significantly different birefringence patterns of TRS starch granules indicated that the arrangement of starch molecules varied between heterogeneous granules and within the same granule. Polarization Colors of Iodine-Stained Heterogeneous Starch Granules. Iodine staining in combination with polarized light microscopy has been used to reveal the structural heterogeneity of starch granules.15,18 The color of iodine-stained starch under polarized light is termed the polarization color.25 TQ and TRS starch granules stained with iodine solution (0.04% I2 and 0.06% KI) were viewed under normal and polarized light (Figure 2). The TQ starch showed a homogeneous blue polarization color at the Maltese cross (Figure 2a,c), while the TRS starch granules exhibited significantly heterogeneous polarization colors. There were three types of polarization colors for TRS individual granules: blue (Figure 2d1), dark red (Figure 2d2), and a dark blue interior with brown exterior (Figure 2d3). The TRS aggregate granules displayed four types of polarization colors: blue (Figure 2e1), fuchsia (Figure 2e2), dark red (Figure 2e3), and a dark blue interior with brown exterior (Figure 2e4). Both the TRS elongated and interior hollow granules exhibited two types of polarization colors: dark red (Figure 2f1,g1) and a dark blue interior with brown exterior (Figure 2f2,g2). Table 1 summarizes the percentages of iodine-stained starch granules with different polarization colors. The TRS starch granules showed significant heterogeneity in polarization color between heterogeneous granules and within the same granule. Most of the TRS individual granules showed a blue polarization color, aggregate granules a dark red polarization color, and elongated and interior hollow granules a dark blue interior with brown exterior polarization color. The present results agree with previous reports that the polarization color of iodine-stained

Figure 1. Microphotographs of starch granules under normal (A−G) and polarized (a−g) light. (A, C, a, c) TQ starch, (B, D−G, b, d−g) TRS starch, (C, D, c, d) individual granule, (E, e) aggregate granule, (F, f) elongated granule, (G, g) interior hollow granule. Scale bar = 10 μm.

granules could be divided into four types: individual, aggregate, elongated, and interior hollow granules according to their morphologies and Maltese crosses (Figure 1D−G,d−g). The individual granules of TRS and TQ starches all had the central hila and one typical Maltese cross on one granule, but there were significant differences in morphology and size. The TRS individual granules were spherical and smaller than the polygonal-shaped TQ individual granules (Figure 1D,d). The TRS aggregate granules were large and voluminous with nonangular rounded bodies and numerous bright Maltese crosses within granules (Figure 1E,e). Under transmission electron microscopy, the large voluminous and nonangular rounded body was observed to consist of many small subgranules. Some of these subgranules at the periphery of the starch fuse together and form a thick band or wall encircling the entire exterior of the starch granule, which prevents the release of subgranules during starch isolation.17 Therefore, the large voluminous and nonangular rounded bodies were called aggregate granules, which were observed to have numerous Maltese crosses (Figure 1e). This agrees with the findings of Wei and his colleagues that the aggregate granule consisted of many subgranules and each subgranule contained a hilum and a Maltese cross.17 The TRS elongated granules displayed different shapes including rods and filaments and could be further classified into three types: full birefringence, partial birefringence, and no birefringence according to their birefringence patterns (Figure 1F,f). The elongated granules with full birefringence patterns had three or more clear Maltese crosses within granules (Figure 1f1). Some of the elongated granules with partial birefringence showed only weak birefringence along the edge of the granules, while others displayed a birefringence pattern consisting of combinations of one or more Maltese crosses and weak to no birefringence on other parts of the granule (Figure 1f2,f3). The third type of 10145

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oriented amylopectin molecules.18 When starch molecules (amylose and amylopectin) are completely radially oriented, iodine-stained starch granules will exhibit a blue polarization color because the amylose−iodine complex generates a stronger color than the amylopectin−iodine complex. When only the amylopectin is oriented radially, the intensity of birefringence will be slightly reduced and iodine-stained starch granules will display a pink polarization color resulting from the amylopectin−iodine complex. When only the amylose is oriented radially, the intensity of birefringence will be markedly diminished and iodine-stained starch granules will display a blue polarization color resulting from the amylose−iodine complex.18 In the present study, all TQ starch granules and most of the TRS individual granules showed a blue polarization color, indicating that these granules contained radially oriented starch molecules. Some TRS aggregate granules exhibited a fuchsia polarization color, indicating that both the amylopectin and amylose were partly radially oriented. As a result of the less symmetrical radial orientation of amylopectin and the randomly oriented amylose, a dark red polarization color was observed in some TRS individual, aggregate, elongated, and interior hollow granules. The dark reddish polarization color has also been observed in some maize amylose-extender (ae) mutant starch granules.18 The polarization colors of a dark blue interior with brown exterior were also observed in TRS starch granules (Figure 2d3,e4,f2,g2). The different colors within a granule indicated that its structure was heterogeneous. This structural heterogeneity within a granule has been also reported in highamylose maize starches.13−15,18 The significantly different polarization colors of the TRS starch granules indicated that heterogeneity in the orientations of amylose and amylopectin existed between heterogeneous granules and within the same granule. Fluorescence Intensities of APTS-Stained Heterogeneous Starch Granules. CLSM photographs of APTS-stained starch granules provide information about the internal structure of starch granules.31 The reducing end of the starch molecule is reacted specifically with APTS leading to a 1:1 stoichiometric ratio for starch molecule labeling. Owing to its smaller size, amylose contains a much higher molar ratio of reducing ends per glucose residue than amylopectin, which leads to a higher by-weight labeling of amylose when constructing a detailed map of the distribution of amylose and amylopectin within starch granules.19 The distribution of amylose and amylopectin within the TQ and TRS starch granules stained with APTS are shown in Figure 3. TQ starch granules showed a strong fluorescent dot in the central hilum (Figure 3A), indicating a high density of amylose in the center of the granule. This phenomenon has also been reported in normal cereal starch granules.17,31 Because of the high content of amylose, the TRS starch granules exhibited a fluorescence too strong for observing the

Figure 2. Microphotographs of iodine-stained starch granules under normal (A−G) and polarized (a−g) light. (A, C, a, c) TQ starch, (B, D−G, b, d−g) TRS starch, (C, D, c, d) individual granule, (E, e) aggregate granule, (F, f) elongated granule, (G, g) interior hollow granule, (c, d1, e1) showing the blue color, (e2) showing the fuchsia color, (d2, e3, f1, g1) showing the dark red color, (d3, e4, f2, g2) showing the dark blue interior with brown exterior. Scale bar = 10 μm.

starch is similar to that observed under normal light for waxy and normal starch and different from that observed under normal light for high-amylose starch. The ae single mutant high-amylose maize starch appears blue under normal light and pink under polarized light.18,25 The explanation of polarization color is based on absorption effects in combination with birefringence.26 It is generally known that the amylose−iodine complex normally shows blue under normal light, but, in fact, the color of the complex depends on the degree of polymerization (DP) of the amylose helix. The color of the amylose−iodine complex changes from brown (DP 21−24) to red (DP 25−29), red−violet (DP 30− 38), blue−violet (DP 37−46), and finally blue (DP > 47).27 In the same manner as amylose, the linear branches of amylopectin, which form a helix, can also develop a red or orange amylopectin−iodine complex. The external chain length is equally important for iodine staining of amylopectin.27−29 It is believed that the radially ordered alignment of starch molecules results in birefringence and the random orientation in no birefringence.30 The intensity of birefringence is not in proportion to the amylopectin content but is positively correlated with the proportion of symmetrically and radially

Table 1. Percentages (%) of Iodine-Stained Starch Granules with Different Polarization Colorsa starches TQ individual granule TRS individual granule aggregate granule elongated granule interior hollow granule a

blue

fuchsia

dark red

dark blue interior with brown exterior

100.0 ± 0.0 c

−b

−

−

46.4 ± 1.8 b 0.3 ± 0.5 a − −

− 23.9 ± 1.4 − −

37.6 ± 0.9 b 38.2 ± 0.5 b 39.9 ± 2.2 b 3.0 ± 0.5 a

16.0 37.6 60.1 97.0

± ± ± ±

2.5 0.6 2.2 0.5

a b c d

Data (mean ± SD) in the same column with different lowercase letters were significantly different (p < 0.05, n = 3). bData were not detected. 10146

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fluorescence. This result suggests that the amylose content, which was largely distributed in the subgranules, was very high in the second type of aggregate granule and would result in a dark red polarization color (Figure 2e3) or a dark blue interior with brown exterior polarization colors (Figure 2e4). The internal structure and amylose distribution of TRS aggregate granules agreed with previous reports on high-amylose rice and maize starch aggregate granules.15,17 For TRS elongated granules, there were three types of amylose distribution (Figure 3D). The first type consisted of three or more regular and polyhedral subgranules. These subgranules were arranged in a line and encircled by a band with the hila of subgranules showing strong fluorescence (Figure 3D1). This type of elongated granule had many clear Maltese crosses (Figure 1f1) and showed a dark red polarization color (Figure 2f1). The second type had no clear subgranule inside, but three or more highly fluorescent clear round dots were arranged in a line inside the granule, which might result from the aberrant formation of subgranules (Figure 3D2). This type of elongated granule had some Maltese crosses (Figure 1f1) and might display a dark red polarization color (Figure 2f1). The third type had an intensely fluorescent interior (Figure 3D3). This type of elongated granule might have only a weak birefringence along the edge of the granule or no birefringence (Figure 1f2−f4) and resulted in a dark blue interior with brown exterior polarization colors (Figure 2f2). The different amylose distribution patterns in elongated starch granules have been reported in high-amylose maize.15,21 For TRS interior hollow granules, irregular granule shapes could be observed and there were three types of amylose distribution (Figure 3E). Some TRS interior hollow granules had a few subgranules adjacent to the encircling band. These subgranules were polyhedral and had central hila. The hila of subgranules and the encircling band of the granule displayed highly strong fluorescence (Figure 3E1). The second type of interior hollow granule showed a brightly fluorescent band encircling the entire circumference and a weakly fluorescent interior that consisted of very small starch molecules (Figure 3E2). The third type of interior hollow granule exhibited a weakly fluorescent band encircling the entire circumference, but the interior of the band displayed very strong fluorescence (Figure 3E3). These results revealed that the formation of the subgranules inside the TRS interior hollow granules was aberrant, which resulted in no birefringence or very weak birefringence in the encircling band (Figure 1g). The strongly fluorescent band indicated a very high amylose content, which resulted in a dark red polarization color (Figure 2g1) or a dark blue interior with brown exterior polarization colors (Figure 2g2). These amylose and amylopectin distributions within the TQ and TRS starch granules (Figure 3) indicate that heterogeneity in amylose and amylopectin distribution existed in all starch granules, which agreed with previous reports.17,19,31 The distribution patterns of amylose and amylopectin (Figure 3) showed that the TQ starch granules were homogeneous, and the TRS starch granules were highly heterogeneous, which agreed with results on high-amylose maize starch.15 As previously reported, the fluorescence intensity of APTSstained starch granules as imaged by CLSM was positively correlated with amylose content.19,32 To compare the difference in amylose content between heterogeneous starch granules, the fluorescence intensities of the optical sections

Figure 3. CLSM optical sections of starch granules stained with APTS. (A) TQ starch, (B−E) TRS starch, (A, B) individual granule, (C) aggregate granule, (D) elongated granule, (E) interior hollow granule. Scale bar = 10 μm.

microstructure under the same conditions of laser power and master gain as the TQ starch (data not shown). Figure 3B−E shows the microstructure and fluorescence distribution of TRS starch granules at a lower laser power. For the TRS individual granules, there were three types of amylose distribution pattern (Figure 3B). For the first type, some TRS individual granules had a slightly smaller size than the TQ starch granules with a bright fluorescent dot in the hilum (Figure 3B1). This type of amylose distribution was similar to that of the individual TQ granule. Starch granules with this type of amylose distribution might display a blue polarization color (Figure 2d1). The second type of TRS individual granule exhibited strong fluorescence evenly over the whole granule (Figure 3B2), indicating a high level of amylose distribution in the whole granule, and might display a dark blue interior with brown exterior polarization colors (Figure 2d3). The third type of TRS individual granule had a significantly smaller size and a larger central strongly fluorescent dot compared with the individual TQ granules. This type of starch also had an intense fluorescence around the granule’s circumference (Figure 3B3), indicating that the amylose was concentrated mainly around the central hilum and the circumference of granule and might result in the dark red polarization color (Figure 2d2). For TRS aggregate granules, the CLSM optical sections clearly revealed that the large voluminous and nonangular rounded bodies consisted of many subgranules and a band encircled the entire circumference of the granule (Figure 3C). There were two types of amylose distribution patterns in the TRS aggregate granules. The first type, consisting of most of the aggregate granules, showed intense fluorescent dots in the hila of subgranules and a bright fluorescence around the circumference of the granules and the subgranules (Figure 3C1), indicating that the amylose was distributed mainly in the central hilum regions of the subgranules and the circumference of the granules and subgranules. This type of aggregate granule might display the blue and fuchsia polarization colors (Figure 2e1,e2). The second type of aggregate granule had such strong fluorescence on the subgranules that the microstructure of the granule could not be clearly viewed under the same viewing conditions as the first type of aggregate granule (data not shown). Figure 3C2 shows the amylose distribution of the second type of aggregate granule using a lower laser power than the first type of aggregate granule. The interior of the subgranules exhibited a uniformly intense fluorescence and the band encircling the entire circumference of granules a weak 10147

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Table 2. Fluorescence Intensities and Gelatinization Temperatures of Starch Granulesa gelatinization temp (°C)c starches TQ individual granule TRS individual granule aggregate granule elongated granule interior hollow granule

fluorescence int (arbitrary units)

b

17.3 ± 6.6 a 26.7 44.4 43.7 63.1

± ± ± ±

15.0 14.7 18.2 16.2

b c c d

Ti

Tm

Te

64.5 ± 0.5 a

72.1 ± 0.2 a

76.3 ± 0.3 a

68.9 ± 0.1 b 72.2 ± 1.1 c 76.5 ± 0.7 d −d

75.1 ± 1.1 b 77.4 ± 1.0 c 80.0 ± 0.1 d −

79.2 ± 1.0 b 82.3 ± 0.5 c 84.6 ± 0.1 d −

Data (mean ± SD) in the same column with different lowercase letters were significantly different (p < 0.05, n = 60 for fluorescence intensity and 3 for gelatinization temperature). bThe intensity of fluorescence was measured in CLSM optical sections of starch granules. cGelatinization temperatures (Ti, initial temperature; Tm, middle temperature; Te, end temperature) were measured by hot stage microscopy. dData were not detected. a

by granule swelling. Although TQ and TRS starches had different morphologies, their crystallinity disruption patterns were similar. During heating, the crystallinity disruption started in the hilum region and was accompanied by a small amount of swelling. With increasing heating temperature, the crystallinity disruption and swelling propagated from the inner hilum to the outer region of the starch granule. At this stage, the crystal structure of the outer area of the granule was still undisrupted. During the last stage of the gelatinization process, the crystallinity of the outer region of the granule was disrupted. These crystallinity disruption patterns for TQ and TRS starches were similar to those of various starches with central hila.34 The gelatinization temperatures of TQ and TRS starch granules with the associated changes in birefringence are presented in Table 2.20 The TRS starches showed significantly higher gelatinization temperatures than the TQ starch. Among the TRS heterogeneous starches, the individual and elongated starch granules had the lowest and highest gelatinization temperatures, respectively. The TRS interior hollow granules exhibited extremely weak or no birefringence, therefore their gelatinization temperature could not be detected in the present study. The gelatinization temperature of starch is related to a variety of factors including granule morphology and size, amylose content, amylopectin structure, and crystallinity type.35,36 In high-amylose starches, the amylose double helices and the amylose−lipid complex require a high temperature and energy to disrupt and therefore give rise to a high gelatinization temperature.37 High-amylose maize starch consists of aggregate and elongated granules. The amylose molecules interact and form antiparallel double helices between the adjacent two subgranules, and the continuous outer layer of these fused granules is composed of more amylose.21 In the present study, the TRS elongated and aggregate granules had higher gelatinization temperatures than the TRS individual granules, indicating that the elongated and aggregate granules had a higher amylose content and gelatinization resistance than the TRS individual granules. These results agreed with the fluorescence intensity of APTS-stained starch granules (Table 2). The swelling of granules was investigated continuously in situ at 5 °C intervals from 40 to 85 °C during heating (Figure 4) and was calculated as the sectional area swelling percentage compared with ungelatinized granules at 40 °C in Table 3. Compared with that at 40 °C, the granule size of TQ and TRS starch did not significantly change at 50 °C and the size started to increase above 55 °C. Below 75 °C, the granule swelling was not significantly different between TQ and TRS starches. The swelling of TQ starch granules increased markedly above 75 °C

were analyzed under the same photographic conditions, which provided no saturation of the detector for every dot in all starch granules in the present study (Table 2). Compared with the TQ starches, TRS starches showed a significantly higher fluorescence intensity, which corresponded with a higher amylose content.6,16 For the TRS heterogeneous starch granules, interior hollow granules and individual granules had the highest and lowest fluorescence intensity, respectively. These results indicated that a significant heterogeneity in amylose content existed between TRS heterogeneous starch granules and that the interior hollow granule and individual granule had the highest and lowest amylose content, respectively. Gelatinization Properties of Heterogeneous Starch Granules. Hot stage microscopy is a sensitive in situ method to investigate the simultaneous behaviors of granule swelling, gelatinization temperature, and crystallinity disruption.20,33,34 The behaviors during TQ and TRS starch gelatinization were investigated using hot-stage microscopy in the present study, and the results are presented in Figure 4. Before gelatinization,

Figure 4. Gelatinization of starch granules under polarized light at different temperatures. (A) TQ individual starch granule; (B, C, D, E) TRS individual, aggregate, elongated, and interior hollow starch granule, respectively. Scale bar = 10 μm.

the starch granule retained its intact morphology as observed under normal light and showed a bright and clear Maltese cross pattern under polarized light. During gelatinization, the birefringence intensity of the granule faded away when the heating temperature rose above the initial gelatinization temperature, and this loss of birefringence was accompanied 10148

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Table 3. Area Swelling Percentages (%) of Starch Granules Measured by Hot Stage Microscopy at Different Temperaturesa TRS temp (°C) 45 50 55 60 65 70 75 80 85

TQ individual granule 100.3 100.5 101.0 102.2 104.8 115.1 183.0 396.1 −b

± ± ± ± ± ± ± ±

0.5 aA 0.5 aA 1.2 aA 1.8 aA 2.9 aA 17.8 aA 38.9 bC 42.0 cE

individual granule 100.2 100.3 101.0 103.0 106.3 112.5 136.7 230.3 −

± ± ± ± ± ± ± ±

0.3 aA 0.4 aA 0.8 aA 3.1 aA 5.5 aA 11.2 aA 25.4 bB 51.2 cD

aggregate granule 100.2 100.3 100.6 102.0 105.0 114.7 131.3 179.6 231.6

± ± ± ± ± ± ± ± ±

0.2 aA 0.3 aA 0.5 aA 1.2 aA 4.6 aA 8.4 aA 14.0 bAB 38.9 cC 62.8 dC

elongated granule 100.1 100.4 100.9 102.6 105.5 110.0 121.5 140.0 157.5

± ± ± ± ± ± ± ± ±

0.3 aA 0.3 aA 0.5 aA 1.8 aA 2.9 aA 3.8 aA 9.0 bAB 20.2 cB 30.3 dB

interior hollow granule 100.2 100.4 100.7 102.3 103.9 108.3 111.7 117.8 127.7

± ± ± ± ± ± ± ± ±

0.2 aA 0.4 aA 1.6 aA 2.2 aA 3.0 aA 3.5 bA 4.4 bA 6.4 cA 11.4 dA

a Data (mean ± SD) in the same column with different lowercase letters and in the same line with different capital letters were significantly different (p < 0.05, n = 50). bData were not detected.

and was significantly higher than that of the TRS starches. Above 75 °C, the swelling of TRS starch granules exhibited significant differences between heterogeneous granules at the same temperature. The swelling of individual and interior hollow granules was the highest and lowest, respectively. Swelling was not measured above 80 °C for TQ starch and TRS individual starch granules and above 85 °C for TRS aggregate, elongated, and interior hollow starch granules because of the deformation, folding, and disruption of the swollen granules at high temperatures. Amylose is considered to contribute to the inhibition of the water absorption and swelling of starch, whereas amylopectin tends to promote these processes. Owing to the decrease of amylopectin content, in high-amylose starch granules, a small quantity of amylose forms the double helical structure through self-interactions. Furthermore, the amylose double helices and the amylose−lipid complex, which only disrupt at high temperature, will strongly inhibit the swelling of high-amylose starch.37 During gelatinization, the starch chains interact in the crystalline and amorphous regions of the highamylose starch granule. The interactions of amylose chains (involving amylose−lipid and amylose−amylose) may result in the formation of new crystallites with varying stability in the amorphous and crystalline regions of the granule. In contrast, the interactions of amylopectin chains do not lead to the formation of new crystallites.38 It has been reported that swelling power is significantly negatively correlated with the amylose content.39,40 In the present study, TRS starch had a significantly lower level of granule swelling than TQ starch above 75 °C, which might result from the high amylose content, the high level of amylose−lipid complex chains, and the interactions of amylose chains that formed the most stable new crystallites in TRS starch. The granule swelling showed significant differences between TRS heterogeneous starch granules. The individual and interior hollow granules had the highest and lowest granule swelling, respectively, which might have resulted from significantly different morphology, amylose content, and amylose distribution. Spatial Distribution of Heterogeneous Starch Granules in Endosperm. The combined use of resin semithin sections in which starch is retained within cells and KI/I2 staining is a powerful in situ technique to observe the kernel shape, starch morphology, and spatial distribution of granules in the endosperm. On the basis of the visual examination of transverse sections at the midregion of 10 mature kernels of TQ and TRS, there was a clear difference in the kernel shape, starch granule morphology, and spatial distribution of starch granules in the endosperm between TQ and TRS (Figures 5

Figure 5. Microphotographs of iodine-stained transverse sections at the midregion of TQ mature kernel. (B−J) Showing starch granule morphology in different regions of endosperm. Scale bar = 200 μm for A and 10 μm for B−J.

and 6). For kernel shape, the thickness of kernel was significantly lower in TRS than in TQ, which agreed with the mature kernel appearance.6 The sections were stained with iodine so that starch granules were specifically stained blue. The degree of filling of the starch was significantly higher in the TQ endosperm than in the TRS endosperm, resulting in the decrease of kernel weight of TRS.6 Regarding the starch granule morphology, TQ showed typical compound starch structure and was homogeneous. The compound starch was composed of many separate subgranules, formed simultaneously within a single amyloplast, but each separate granule had a central hilum (Figure 5). During grain isolation, the compound starch can be ground into separate individual granules. TRS starch showed significant heterogeneity in its morphology as compound, aggregate, elongated, and interior hollow starches. The compound starch can be broken up into individual starch granules, while aggregate granules can remain intact with the surrounding band (Figure 6). 10149

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at the central region of the endosperm and propagates outward to the outer endosperm cells. Consequently, the peripheral endosperm cells are the final parts of the endosperm to be filled with starch granules. This process is completed about 1 month after the initiation of starch granule formation in the central region of the endosperm.41 This would imply that the greatest level of heterogeneity occurred in TRS starch granules that were deposited late during the development of the kernel, and that the level of heterogeneity was less in granules formed during the early development of the kernel. Cereal storage starches are semicrystalline granules consisting of the two main components: amylose and amylopectin. Their biosynthesis is a complex system involving four classes of enzyme: ADP-glucose pyrophosphorylase, starch synthase, SBE, and starch debranching enzyme. The granule-bound starch synthase is essential for amylose biosynthesis, while amylopectin is synthesized by the combined actions of several isoforms of starch synthase, SBE, and starch debranching enzyme.42 The SBEs play a pivotal role in amylopectin biosynthesis and occur in cereals in three classes: SBE I, IIa, and IIb.8 SBE I plays a role in the formation of long chains of amylopectin, SBE IIb generates short chains, and SBE IIa partially, but not fully, supports the functions of SBE I and IIb.6 The expression of starch-related enzymes varies dramatically between the early and late stages of endosperm development.43 The granule-bound starch synthase I (GBSS I) is the most important enzyme for amylose biosynthesis in the endosperm. The mRNA of GBSS I increases with the development of rice endosperm,44 and the activities of both SBE I and IIb are inhibited in the middle and late stages of TRS kernel development.6 The inhibition of SBE I/IIb not only increases amylose content but also changes the structure of starch granules, leading to a broad population of morphological types. The spatial distribution of heterogeneous starch granules has suggested that the individual granule (compound starch) was synthesized at the early stage of endosperm development, the aggregate and elongated granules at the middle stage, and the interior hollow granules at the late stage. The formation and spatial distribution of these heterogeneous starch granules result from changes in the activities of SBEs during kernel development. In conclusion, a range of microscopic techniques have been used to observe and characterize the morphology and structure of starch granules isolated from the mature kernels of highamylose rice (TRS) and its wild-type rice (TQ). The mature kernels have been mapped in situ using light microscopy to identify the spatial distribution of starch granules in the endosperm. These studies have revealed that TQ starch granules were homogeneous in their morphology, structure, and spatial distribution in the endosperm. In contrast, TRS starch showed significant heterogeneity in morphology consisting of individual, aggregate, elongated, and interior hollow granules. The polarization colors of iodine-stained starch granules and fluorescence intensities of APTS-stained starch granules indicated that the individual and interior hollow granules had the lowest and highest amylose contents, respectively, among the TRS heterogeneous starch granules. The results of hot-stage microscopy showed that the individual and interior hollow granules had the lowest and highest resistance to heating, respectively. The kernel sections revealed that the individual granules existed mainly in the middle of the endosperm, the interior hollow granules in the subaleurone layer cells, the elongated granules in the peripheral starchy endosperm cells adjacent to the subaleurone layer, and the

Figure 6. Microphotographs of iodine-stained transverse sections at the midregion of TRS mature kernel. (B) Showing the distribution of heterogeneous starch granules in subaleurone layer and starchy endosperm cells, (C−F) showing interior hollow starch granule in region I of endosperm, (G−I) showing elongated starch granule in region II of endosperm, (J, K) showing aggregate starch granule in region III of endosperm, (L) showing individual starch granule in region IV of endosperm. Scale bar = 200 μm for A, 100 μm for B, and 10 μm for C−L.

Regarding the spatial distribution of starch, the compound starch was homogeneously distributed in the whole TQ endosperm, except that the central endosperm cells were packed more tightly than the surrounding endosperm cells (Figure 5). TRS heterogeneous starch granules had a significant regional distribution in the endosperm (Figure 6). The interior hollow starch granules (Figure 6C−F) were mainly distributed in the subaleurone layer (Figure 6, region I), the elongated granules (Figure 6G−I) in the peripheral starchy endosperm cells adjacent to the subaleurone layer (Figure 6, region II), the aggregate granules (Figure 6J,K) in the starchy endosperm cells between the subaleurone layer and the central region of kernel (Figure 6, regions II and III), and the compound starch (Figure 6L) in the central regional of the kernel (Figure 6, region IV). The spatial distribution of starch granules was mapped within the kernel. The present study suggests that the level of heterogeneity of the TRS starch granules in the endosperm varied remarkably between different regions of the endosperm and became more heterogeneous from the inner to the outer endosperm. The highest level of heterogeneity was observed in the peripheral starchy endosperm adjacent to the subaleurone layer (Figure 6, region II). It is necessary to explain the reason for this spatial distribution of heterogeneous starch granules in the endosperm. In rice, the formation of starch granules starts 10150

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(10) Jiang, H. X.; Campbell, M.; Blanco, M.; Jane, J. L. Characterization of maize amylose-extender (ae) mutant starches: Part II. structures and properties of starch residues remaining after enzymatic hydrolysis at boiling-water temperature. Carbohydr. Polym. 2010, 80, 1−12. (11) Regina, A.; Kosar-Hashemi, B.; Ling, S.; Li, Z. Y.; Rahman, S.; Morell, M. Control of starch branching in barley defined through differential RNAi suppression of starch branching enzyme IIa and IIb. J. Exp. Bot. 2010, 61, 1469−1482. (12) Kim, K. S.; Hwang, H. G.; Kang, H. J.; Hwang, I. K.; Lee, Y. T.; Choi, H. C. Ultrastructure of individual and compound starch granules in isolation preparation from a high-quality, low-amylose rice, Ilpumbyeo, and its mutant, G2, a high-dietary fiber, high-amylose rice. J. Agric. Food Chem. 2005, 53, 8745−8751. (13) Wellner, N.; Georget, D. M. R.; Parker, M. L.; Morris, V. J. In situ Raman microscopy of starch granule structures in wild type and ae mutant maize kernels. Starch/Stärke 2011, 63, 128−138. (14) Liu, D.; Parker, M. L.; Wellner, N.; Kirby, A. R.; Cross, K.; Morris, V. J.; Cheng, F. Structural variability between starch granules in wild type and in ae high-amylose mutant maize kernels. Carbohydr. Polym. 2013, 97, 458−468. (15) Cai, C. H.; Zhao, L. X.; Huang, J.; Chen, Y. F.; Wei, C. X. Morphology, structure and gelatinization properties of heterogeneous starch granules from high-amylose maize. Carbohydr. Polym. 2014, 102, 606−614. (16) Wei, C. X.; Xu, B.; Qin, F. L.; Yu, H. G.; Chen, C.; Meng, X. L.; Zhu, L. J.; Wang, Y. P.; Gu, M. H.; Liu, Q. Q. C-type starch from highamylose rice resistant starch granules modified by antisense RNA inhibition of starch branching enzyme. J. Agric. Food Chem. 2010, 58, 7383−7388. (17) Wei, C. X.; Qin, F. L.; Zhu, L. J.; Zhou, W. D.; Chen, Y. F.; Wang, Y. P.; Gu, M. H.; Liu, Q. Q. Microstructure and ultrastructure of high-amylose rice resistant starch granules modified by antisense RNA inhibition of starch branching enzyme. J. Agric. Food Chem. 2010, 58, 1224−1232. (18) Evans, A.; McNish, N.; Thompson, D. B. Polarization colors of lightly iodine-stained maize starches for amylose-extender and related genotypes in the W64A inbred line. Starch/Stärke 2003, 55, 250−257. (19) Blennow, A.; Hansen, M.; Schulz, A.; Jørgensen, K.; Donald, A. M.; Sanderson, J. The molecular deposition of transgenically modified starch in the starch granule as imaged by functional microscopy. J. Struct. Biol. 2003, 143, 229−241. (20) Konik-Rose, C. M.; Moss, R.; Rahman, S.; Appels, R.; Stoddard, F.; McMaster, G. Evaluation of the 40 mg swelling test for measuring starch functionality. Starch/Stärke 2001, 53, 14−20. (21) Jiang, H. X.; Horner, H. T.; Pepper, T. M.; Blanco, M.; Campbell, M.; Jane, J. L. Formation of elongated starch granules in high-amylose maize. Carbohydr. Polym. 2010, 80, 533−538. (22) Yamamori, M.; Quynh, N. T. Differential effects of Wx-A1,-B1 and-D1 protein deficiencies on apparent amylose content and starch pasting properties in common wheat. Theor. Appl. Genet. 2000, 100, 32−38. (23) Gallant, D. J.; Bouchet, B.; Baldwin, P. M. Microscopy of starch: evidence of a new level of granule organization. Carbohydr. Polym. 1997, 32, 177−191. (24) Pérez, S.; Bertoft, E. The molecular structures of starch components and their contribution to the architecture of starch granules: a comprehensive review. Starch/Stärke 2010, 62, 389−420. (25) Seckinger, H. L.; Wolf, M. J. Polarization colors of maize starches varying in amylose content and genetic background. Starch/ Stärke 1966, 18, 1−5. (26) Evans, A.; Thompson, D. B. Resistance to α-amylase digestion in four native high-amylose maize starches. Cereal Chem. 2004, 81, 31−37. (27) John, M.; Schmidt, J.; Kneifel, H. Iodine-maltosaccharide complexes: relation between chain-length and colour. Carbohydr. Res. 1983, 119, 254−257. (28) Knutson, C. A. Evaluation of variations in amylose-iodine absorbance spectra. Carbohydr. Polym. 1999, 42, 65−72.

aggregate granules in the starchy endosperm cells between the subaleurone layer and the middle of the endosperm. These results suggest that the morphology and structure of TRS starch granules and their spatial distribution in endosperm were all heterogeneous. This heterogeneity might have resulted during kernel development.

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AUTHOR INFORMATION

Corresponding Author

*College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, China. Tel: +86 514 87997217. Email: [email protected]. Funding

This study was financially supported by grants from the Ministry of Science and Technology of China (2012CB944803), the National Natural Science Foundation of China (31270221), the Qing Lan Project of Jiangsu Province, the Talent Project of Yangzhou University, the Innovation Program for Graduates of Jiangsu Province (CXZZ13_0895), and the Priority Academic Program Development of Jiangsu Higher Education Institutions. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED APTS, 8-amino-1,3,6-pyrenetrisulfonic acid; CLSM, confocal laser scanning microscope; RS, resistant starch; SBE, starch branching enzyme; TQ, Te-qing (wild type rice cultivar); TRS, transgenic RS rice line

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REFERENCES

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