Characterization of Grain Quality and Starch Fine Structure of Two

Apr 29, 2016 - Rice is one of the most widely consumed cereal crops worldwide. ... contain low grain amylose content but exhibit good sensory properti...
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Characterization of Grain Quality and Starch Fine Structure of Two Japonica Rice (Oryza Sativa) Cultivars with Good Sensory Properties at Different Temperatures during the Filling Stage Changquan Zhang,†,∥ Lihui Zhou,†,‡,∥ Zhengbin Zhu,§ Huwen Lu,† Xingzhong Zhou,† Yiting Qian,† Qianfeng Li,† Yan Lu,† Minghong Gu,† and Qiaoquan Liu*,† †

Key Laboratory of Plant Functional Genomics of the Ministry of Education, Co-Innovation Center for Modern Production Technology of Grain Crops, College of Agriculture, Yangzhou University, Yangzhou 225009, China ‡ Jiangsu High Quality Rice Research and Development Center, Institute of Food Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China § Suzhou Seed Administration Station, Suzhou 215011, China S Supporting Information *

ABSTRACT: Temperature during the growing season is a critical factor affecting grain quality. High temperatures at grain filling affect kernel development, resulting in reduced yield, increased chalkiness, reduced amylose content, and poor milling quality. Here, we investigated the grain quality and starch structure of two japonica rice cultivars with good sensory properties grown at different temperatures during the filling stage under natural field conditions. Compared to those grown under normal conditions, rice grains grown under hot conditions showed significantly reduced eating and cooking qualities, including a higher percentage of grains with chalkiness, lower protein and amylose contents, and higher pasting properties. Under hot conditions, rice starch contained reduced long-chain amylose (MW 107.1 to 107.4) and significantly fewer short-chain amylopectin (DP 5−12) but more intermediate- (DP 13−34) and long- (DP 45−60) chain amylopectin than under normal conditions, as well as higher crystallinity and gelatinization properties. KEYWORDS: rice, Oryza sativa, eating and cooking quality, starch fine structure, climate change, field

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INTRODUCTION Rice is one of the most widely consumed cereal crops worldwide. Along with increasing yields, improving rice grain quality is one of the most important goals of rice breeders to meet market demands.1 Because up to 90% of polished rice grains are composed of starch, the physicochemical properties and fine structure of starch greatly affect grain quality as well as rice-flour quality.2 During grain filling, the field environment (in particular, air temperature) has a significant effect on both grain composition and the structure and properties of the starch.3,4 As a result, the specific effects of the field environment are becoming an increasing concern.5−8 Grain quality in rice is a complex trait with various determinants, including physical appearance, eating and cooking qualities (ECQ), and nutritional value. ECQ is the most important factor for consumers and is associated with characteristics such as glossiness, flavor, and stickiness.9 Some automatic testing systems (for example, the rice Taste Analyzer) are employed to measure the quality of milled rice and thus indirectly assess the taste value of cooked rice. In addition, several other characteristics are also examined, mainly to determine the physicochemical properties of rice starch, including hydration, gelatinization, volume expansion, and digestion properties.2 Previously established physicochemical characteristics, such as apparent amylose content (AAC), gelatinization temperature (GT), and gel consistency (GC), are used to evaluate the ECQ of rice.10 Starch is a homopolymer of © 2016 American Chemical Society

glucosyl units consisting of amylose and amylopectin molecules, which are assembled together to form a semicrystalline starch granule.11−13 Important starch property parameters, such as pasting viscosity characteristics, gel texture, thermal and retrogradation properties, and amylose and amylopectin fine structure, have been established to precisely evaluate the enduse quality of cooked rice and starch-based foods.14,15 In addition to genotypic differences, air temperature during the growing season, especially grain filling, is a critical factor affecting grain quality. Previous experiments using growth chambers showed that high temperatures at the grain-filling stage affect kernel development, resulting in reduced yield, increased chalkiness, reduced amylose content, and poor milling quality.4,15 Starch properties are also quite different among different climates and growing seasons.16−18 The earth’s average surface air temperature is predicted to increase by 1.8− 4.5 °C by the end of the century. Thus, there is increasing interest in the impact of temperature on rice production and grain quality.19−21 Recent greenhouse studies (controlledtemperature studies) have shown that the air temperature during the grain-filling stage has a crucial effect on the characteristics of endosperm starch in rice.22 However, Received: Revised: Accepted: Published: 4048

January 9, 2016 April 26, 2016 April 29, 2016 April 29, 2016 DOI: 10.1021/acs.jafc.6b00083 J. Agric. Food Chem. 2016, 64, 4048−4057

Article

Journal of Agricultural and Food Chemistry

Figure 1. Temperatures during the grain-filling stage in 2013 and 2014. HT and LT indicate the highest and lowest temperatures in a day, respectively. Arrows indicate the heading date of the two cultivars in two years.

2013 and 2014, respectively. All growing conditions including field management practices were similar during the two growing seasons except for temperature during grain filling. The air temperature in September, especially in the daytime, was higher in 2013 than in 2014 (Figure 1). The average temperature was approximately 30 °C in 2013 but only approximately 28 °C in 2014. The seeds were harvested at maturity from ten plants from the middle of each plot and airdried. Data from each sample represent the mean from three plots, and the final four samples (N-2013 [H], N-2014 [L], J2013 [H], and J-2014 [L]) represent data from cultivars Nanjing46 (N) and Jia58 (J) grown in 2013 and 2014, respectively. [H] and [L] represent high and low temperature in 2013 and 2014, respectively. Flour and Starch Preparation. After at least three months of storage, the seeds were dehusked with a rice huller (model SY88-TH, Sangyong, Korea) and polished using a grain polisher (Kett, Tokyo, Japan). The size and weight of the polished rice were then measured, and the appearance of the milled rice (percentage of grains with chalkiness, PGWC) was recorded with a rice-grain appearance analysis system (Wseen, China). Milled rice samples were stored in sealed bags under refrigeration (4 °C) until analysis. The polished rice was ground into flour in a mill (FOSS 1093 Cyclotec Sample Mill, Sweden) with a 0.5 mm screen and starch was isolated as described by Zhang et al.24 Taste Value and Cooking Properties. The taste value of milled rice was determined with an RCTA-11A Taste Analyzer (Satake, Japan). To determine the cooking properties of the rice, we washed 30 g of milled rice in a stainless-steel mesh container, transferred it to a 50 mL aluminum box containing 40 mL of distilled water, and cooked it in an electric rice cooker (model Z06YA3-S2, Supor, China). After 20 min of equilibrium, the sensory properties of the cooked rice were evaluated with an STA-1A 18 rice sensory analyzer (Satake, Japan). Physicochemical Characteristics. The apparent amylose content of milled rice flour was determined using an iodine colorimetric method,25 and the true amylose content (AC) was calculated based on gel permeation chromatography (GPC) analysis. The total starch content of the flour was determined using a total starch assay kit (K-TSTA, Megazyme; Wicklow, Ireland). The gel consistency and gelatinization temperature were measured following the method of Tan et al.26 The protein content was determined by nitrogen combustion with a

extrapolating data from greenhouse studies to large-scale longterm rice cultivation in the field is unlikely to be very accurate.22 Therefore, additional studies about the effects of field conditions on rice grain quality are required to ensure the applicability of the experimental data to field production.7 Increases in temperature as a result of global warming have been threatening rice production in many countries over the past few decades.21,22 In China, more emphasis has recently been directed toward the stability of good ECQ rice under different growing conditions in the field. A study of 27 rice cultivars grown in cold and hot locations yielded similar findings as did nonfield studies in terms of starch composition and thermal properties.23 Nevertheless, little is known about the effects of different growing conditions on grain quality or the physiochemical and structural characteristics of endosperm starch under field conditions, especially in years with extreme weather events. In light of extreme climate change, understanding the magnitude of the effects of field environmental condition on starch properties will provide valuable clues for breeders and farmers about the relationship between grain quality and the structure and functional properties of starch. In the present study, the grain quality and starch structure of two japonica rice cultivars with good sensory properties were determined and carefully compared. Experiments were performed in two successive years (2013−2014) under natural field conditions on an experimental farm in Suzhou, China. Fortunately, the air temperature was quite different during the rice grain-filling stage between the two years, allowing the thorough analysis of the effect on grain quality and starch fine structure.

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MATERIALS AND METHODS Rice Samples and Growth Condition. Two recently released rice cultivars, Oryza sativa L. ssp. japonica cv. Nanjing46 and Jia58, were used in this study; both contain low grain amylose content but exhibit good sensory properties. During the growing season from May to October of 2013 and 2014, the two cultivars were planted in the same field at the experimental farm of Yangzhou University in Suzhou city, Jiangsu, China. The plots were arranged in a randomized block pattern with three replications. Each plot consisted of ten rows with ten plants per row. The flowering date was similar for both cultivars in the same year. The initial flowering date for Nanjing46 was September 2 and 6 in 2013 and 2014, respectively, while that for Jia58 was September 5 and 8 in 4049

DOI: 10.1021/acs.jafc.6b00083 J. Agric. Food Chem. 2016, 64, 4048−4057

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Journal of Agricultural and Food Chemistry Table 1. Shape, Weight, and Chalkiness of Rice Grains from Each Cultivar cultivarsa Nanjing46-2013 [H] Nanjing46-2014 [L] Jia58-2013 [H] Jia58-2014 [L]

grain length (mm)

grain width (mm)

grain thickness (mm)

ratio of grain length to width

weight of 1000 grains of polished rice (g)

percentage of grains with chalkiness (%)

4.8 ± 0.1*b

2.8 ± 0.1*

2.1 ± 0.1*

1.69 ± 0.1*

21.3 ± 0.2*

42.6 ± 3.6*

4.9 ± 0.1*

3.0 ± 0.1**

2.1 ± 0.1*

1.64 ± 0.1**

22.9 ± 0.2**

12.3 ± 1.8**

4.9 ± 0.2* 4.9 ± 0.1*

2.9 ± 0.1* 3.0 ± 0.1*

2.1 ± 0.1* 2.1 ± 0.1*

1.68 ± 0.1* 1.64 ± 0.0**

21.5 ± 0.2* 21.9 ± 0.2*

19.8 ± 2.0* 12.5 ± 1.2**

a

[H] and [L]: hot (high temperature) and normal (low temperature) season during the grain-filling stage, respectively. bValues with the same number of asterisks in a column for each cultivar are not significantly different (p < 0.05).

X-ray Powder Diffraction. X-ray powder diffraction (XRD) analysis of starch was carried out on a D8 ADVANCE type X-ray diffractometer (D8, Bruker, Germany), and the relative crystallinity of the starch was measured as described by Wei et al.31 ATR−FTIR. The external region structure of native starch was measured using attenuated total reflectance−Fourier transform infrared (ATR−FTIR) on a Varian 7000 FTIR spectrometer with a DTGS detector equipped with an ATR single reflectance cell containing a germanium crystal (45° incidence angle; PIKE Technologies). Spectra were corrected by a baseline in the region from 1200 to 800 cm−1 before deconvolution was applied using Resolutions Pro. Absorbance values at 1047 and 1022 cm−1 were extracted from the spectra after water subtraction, baseline correction, and deconvolution. Statistical Analysis. For sample characterization, at least two replicate measurements were performed unless otherwise specified. All data represent the means ± standard deviation (means ± SD) of three plots. The results were analyzed using the Student’s t test to examine differences. Results with a corresponding probability value of p < 0.05 were considered to be statistically significant.

nitrogen determinator (FOSS TECATOR Kjeltec230) according to AOAC standard method 990.03.27 The moisture content was measured according to AACC Air Oven Method 4419.21.28 The pasting properties of rice flour or isolated starch were investigated with a Rapid Visco-Analyzer (RVA) (Techmaster, Newport Scientific; Warriewood, Australia) according to the methods of Zhu et al.29 Starch gelatinization and retrogradation temperatures were measured with a differential scanning calorimeter (DSC 200 F3, Netzsch Instruments NA LLC; Burlington, MA) according to the methods of Zhang et al.24 All tests were performed in triplicate. Morphology of Isolated Starch. The structure of the starch granules was observed under an environmental scanning electron microscope (SEM, Philips XL-30). Each starch sample was suspended in ethanol and applied to an aluminum stub using double-sided adhesive tape. The starch samples were coated with gold using a sputter coater, observed, and photographed. To investigate the size distribution, we calculated granular size (diameter) using ImageJ software (http://rsbweb.nih.gov/ij/) based on the SEM image. More than 300 starch granules were analyzed per sample. Determination of Starch Swelling Power. Swelling power was determined in triplicate using 100 mg of native starch by heating starch-water slurries in a water bath at 55, 65, 75, 85, and 95 °C as described by Li and Yeh.30 Fine Structure of Starch. Purified rice starch was debranched with isoamylase (EC3.2.1.68, E-ISAMY, Megazyme), and the molecular weight distribution of debranched starch was determined by GPC with a PL-GPC 220 system (Polymer Laboratories Varian, Inc.; Amherst, MA). The PLGPC 220 system included three columns (PL110-6100, -6300, and -6525) and a differential refractive index detector. Standard dextrans of known molecular weights (2800, 18 500, 111 900, 410 000, 1 050 000, 2 900 000, and 6 300 000) were used for column calibration and, on the basis of the standards, we determined that the relative molecular weight (molecular size) calculated. The experiments were performed twice. The debranched starch was also quantitatively analyzed using a high-performance anion-exchange chromatograph (HPAEC; Thermo ICS-5000, Thermo Corp., Sunnyvate, CA) equipped with a pulsed amperometric detector, a guard column, a CarboPacTM PA200 analytical column, and an AS-DV autosampler. Eluent A was 150 mM NaOH, and eluent B was 150 mM NaOH containing 500 mM sodium acetate. The gradient program was as follows: 35% of eluent B at 0 min, 35% at 2 min, 60% at 17 min, 80% at 40 min, and 80% at 45 min. The separations were carried out at 25 °C with a flow rate of 0.5 mL/min. The concentration of debranched starch and CSCA products was 1 mg/mL in 0.1 M sodium acetate solution.

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RESULTS AND DISCUSSION Comparison of Grain Appearance between the Two Growing Seasons. We first examined the appearance of the polished rice grains, revealing differences in grain width and the ratio of grain length to width in both cultivars in different seasons (Table 1 and Figure 2). Grain width, especially in Nanjing46, decreased in 2013 compared with the width in 2014. Both cultivars showed a significant decrease in the ratio of grain length to width in the hot season, explaining the decrease in 1000 grain weight in Nanjing 46 in 2013. The PGWC was then analyzed, revealing a much higher PGWC in both cultivars in 2013 compared to those in 2014 (Figure 1 and Table 1). Compared with Jia58, grains of Nangjing46 grown in the hot season (N-2013 [H]) were more severely chalky than those grown at normal season (N-2014 [L]), suggesting that grains of Nanjing 46 are more sensitive to hot growing conditions than Jia58 seeds during kernel development. Indeed, several studies have reported that high temperatures during the grain-filling stage result in grains with varying degrees of chalkiness and weight reductions.32,33 These results help confirm the notion that high temperature during grain filling is one of the most serious factors affecting grain development. Physicochemical Characteristics of Milled Rice. To examine the effect of high temperature on rice ECQ formation, 2 we investigated and compared the major components and physicochemical characteristics of flour obtained in the two growing seasons. All four rice-flour 4050

DOI: 10.1021/acs.jafc.6b00083 J. Agric. Food Chem. 2016, 64, 4048−4057

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4051

[H] and [L]: hot (high temperature) and normal (low temperature) season during the grain-filling stage, respectively. bData obtained from GPC analysis. cValues with the same number of asterisks in a column of the same cultivar are not significantly different (p < 0.05). a

74.12 ± 0.85* 75.59 ± 0.82** 74.50 ± 0.71* 75.10 ± 0.71* 4.83 ± 0.55* 4.67 ± 0.41* 8.70 ± 0.20* 8.40 ± 0.26** 11.57 ± 0.55* 13.54 ± 0.30** 84.50 ± 0.59* 86.22 ± 0.69** 7.25 ± 0.07* 6.70 ± 0.14** 14.10 ± 0.14* 14.25 ± 0.09*

14.05 ± 0.21* 15.25 ± 0.07**

77.12 ± 0.58** 75.50 ± 0.71** 4.50 ± 0.54* 9.07 ± 0.15** 10.75 ± 0.11** 6.10 ± 0.14** 14.05 ± 0.12*

Nanjing462013 [H] Nanjing462014 [L] Jia58-2013 [H] Jia58-2014 [L]

86.67 ± 0.44**

84.25 ± 0.75* 7.45 ± 0.07* 13.95 ± 0.17*,c

15.80 ± 0.28**

73.09 ± 0.92* 72.50 ± 0.71* 4.67 ± 0.54* 9.50 ± 0.20* 9.54 ± 0.17*

total starch content (%, w/w) protein content (%, w/w) moisture content (%, w/w) cultivarsa

Table 2. Physicochemical Characteristics and Cooking Properties of Milled Rice

samples had a similar moisture content; however, the protein content (PC) was significantly higher in the 2013 samples (Table 2). This result is consistent with the previous finding that high temperatures at the grain-filling stage lead to increased grain PC.4 The data of total starch content (TSC) assaying showed that grains from the hot season contained a significantly lower TSC accompanied by an increase in PC (Table 2). The apparent amylose contents of both cultivars was higher than the real amylose content according to GPC analysis, with the amylopectin−iodine complexes absorbing similar wavelengths to the amylose−iodine complexes in colorimetry (Table 2). In contrast, the AAC of both cultivars was significantly lower in 2013 than 2014. We also estimated the true AC of the starch using GPC analysis, revealing lower values in the hot season in 2013 than in 2014. Indeed, high-temperature-ripened rice grains exhibit reduced amylose levels, perhaps due to the repressed expression of several starch synthases at high temperatures, such as granule-bound starch synthase I (GBSSI).34 We carried out SDS-PAGE analysis to investigate GBSSI levels in all four rice samples. Both Nanjing46 and Jia58 grown in 2013 exhibited a lower GBSSI level compared to that in rice grown in 2014 (data not shown). We also evaluated the GC and GT of rice flour from the two varieties (Table 2). Nanjing46 exhibited a relatively higher GC than Jia58 in both years, possibly due to the relatively low AC. Both cultivars had significantly higher GC in 2013 than 2014, possibly due to the relatively low AC caused by the hot season. However, no significant differences in GT were detected between samples from both years. Rice Cooking Properties. We initially evaluated the cooking properties of milled rice using the NIR (near-infrared reflectance) technique. The taste value of milled rice of N-2013 [H] was much lower than that of N-2014 [L] and in J-2013 [H] compared to that of J-2014 [L] (Table 2). To evaluate the ECQ more precisely, we used a rice sensory analyzer to evaluate the palatability of cooked rice. The palatability of cooked rice of both cultivars was significantly higher in 2014

apparent amylose content (%, w/w)

true amylose contentb (%, area ratio)

Figure 2. Appearance of polished grains from cultivars Nanjing46 (A,B) and Jia58 (C,D). Panels A and C show grains from the hot season in 2013, and panels B and D show grains from the normal season in 2014.

14.90 ± 0.14*

gel consistence (cm)

gelatinization temperature (ASV)

taste of milled rice (value)

taste of cooked rice (value)

Journal of Agricultural and Food Chemistry

DOI: 10.1021/acs.jafc.6b00083 J. Agric. Food Chem. 2016, 64, 4048−4057

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Journal of Agricultural and Food Chemistry

protein disulfide bonds by adding 5 mmol L−1 DTT to the flour samples (Figure 3C). When DTT was added to the flour− water suspension, the values in the pasting curve decreased for all samples, but the differences observed for each cultivar grown in different years were similar to those of water-treated samples. We also analyzed the pasting curves of isolated starch. All pasting characters of starch samples grown in 2013 were significantly higher than those in the 2014 samples (Figure 3D), which is quite different from the data for rice flour. Although similar trends were observed in the pasting curves, the change in the peak time for RVA characters was greater for Nanjing 46 than for Jia58. Taken together, these results indicate that the differences in pasting properties of flour between the two growing seasons were not due to differences in endogenous enzymes (such as αamylase) or protein structures but were instead due to differences in starch composition and fine structure. Morphological Structure and Swelling Power of Starch. We compared the morphology of isolated starch from both cultivars and growing conditions via scanning electron microscopy. The starch granules of all four samples had similar morphologies; that is, polyhedral and irregular shapes with sharp angles and edges (Figure 4A−D). However, the starch granules from Nanjing46 grown in 2013 (N-2013 [H]) appeared to contain more round granules than those grown in 2014 (N-2014 [H]) (Figure 4A,B), and the granule size also differed between the two seasons. Whereas all starch

than in 2013. Chun et al. reported that the palatability of rice tends to decrease in response to an increase in ripening temperatures above 28 °C in the greenhouse.8 Such differences might be due to the strong change in PGWC, as observed in the present study (Table 1); several studies have shown that the palatability of cooked rice decreases with increasing proportion of chalky rice, as revealed in sensory evaluation tests.8,33 Pasting Properties of Rice Flour and Starch. The pasting properties revealed by RVA measurements reflect changes in the apparent viscosity of a sample during heating and cooling in sufficient water, which predicts the texture of cooked rice. As shown in Figure 3A, the viscosity of rice flour

Figure 3. Rapid viscosity profiles of the flour (A−C) and starch (D). Panels A−C: RVA of flour with 10% (w/v) solids in water (A), in water with 0.5 mM AgNO3 (B), and in water with 5 mM DTT (C), respectively. Panel D: RVA of purified starch with 7% (w/v) solids in water. N-2013 [H], N-2014 [L], J-2013 [H], and J-2014 [L] represent samples from cultivars Nanjing46 (indicated as N) and Jia58 (indicated as J) grown in 2013 with a hot season (high temperature) [H] and 2014 with a normal season (low temperature) [L], respectively.

from the two seasons was quite different, but the patterns of change in apparent viscosity exhibited similar tendencies. However, the peak, trough, breakdown, final viscosities, and pasting temperature were clearly higher and the decrease during cooling was lower in flour from rice grown in the hotter year, 2013 (Supplementary Table S1). Many endogenous factors other than starch, such as αamylase, starch granule-associated proteins, and the fine structure of amylose and amylopectin may affect the RVA pasting curve.35 We used silver nitrate (AgNO3) as an αamylase inhibitor to determine whether the presence of endogenous amylase activity resulted in the differences in rice-flour pasting properties between years. When 0.5 mM AgNO3 solution was used instead of water, all of the viscosity values increased, as the α-amylase in rice flour was inhibited by the silver nitrate (Supplementary Table S1), with both cultivars showing similar pasting curves compared to water-treated samples (Figure 3B). To examine the effects of protein structure on the pasting properties of rice flour, we disrupted

Figure 4. Scanning electron microscopy micrographs of purified starch. Panels A and B show starch from Nanjing46 grown in 2013 and 2014, respectively. Panels C and D show starch from Jia58 grown in 2013 and 2014, respectively. Panels E and F show the swelling powers of starch for Nanjing46 and Jia58, respectively. N-2013 [H], N-2014 [L], J-2013 [H], and J-2014 [L] represent samples from the cultivars Nanjing46 (indicated as N) and Jia58 (indicated as J) grown in 2013 with a hot season (high temperature) [H] and 2014 with a normal season (low temperature) [L], respectively. 4052

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long B chains with higher molecular weight molecules.37 The relative area ratio of the Ap1-to-Ap2 fraction can be used as an index of the extent of amylopectin branching, the higher the ratio, the higher the branching degree.38 The data from GPC analysis indicated that each sample contained similar amylopectin levels. However, the Ap1-to-Ap2 ratio differed between the two growing seasons. As shown in Table 3, rice starch from 2013 tended to have a lower Ap1-to-Ap2 ratio, suggesting that cultivars grown in 2013 (at higher temperatures) might generate fewer A and short B1 chains. In GPC analysis, the third fraction (Am) represents the amylose fraction and consists of two peaks (Am1 and Am2), which represent the relatively short and longer chains, respectively.39 The molecular weight distributions of the Am fraction showed significant difference between cultivars grown in different years (Figure 5C,D). The Am1-to-Am2 ratio was higher for samples grown in 2013 (Table 3), suggesting that starch from rice grown at high temperatures contains more short-chain amylose than that obtained at lower temperatures. In contrast, as shown in Figure 5E,F, rice grains harvested in 2014 tended to contain more long-chain amylose (MW 107.1 to 107.4), which has not been reported previously. We further analyzed the debranched amylopectin fractions by HPAEC. As shown in Figure 6, alterations in the degree of polymerization (DP) for the peak chain length distribution of the same cultivar between the two growing seasons exhibited similar tendencies. The proportion of short chains ranging from DP 5 to DP 12 was obviously lower in 2013 than 2014, which is in good agreement with the above results of GPC analysis, whereby the Ap1-to-Ap2 ratio was lower in 2013 than 2014. However, the relative amounts of intermediate (DP 13−34) and long chains (DP 45−60) of amylopectin were significantly higher in 2013. These results indicate that the amylopectin molecules in rice grown in 2013 had a more homogeneous mass distribution than those from 2014. A similar result was reported in wheat and rice; however, the difference in DP 45− 63 has not previously been reported.8,40 A number of studies have shown that rice grain development was impaired under high-temperature stress. Transcriptomic41 and proteomic42 data suggest that high temperatures mainly affect the expression of genes related to oxidation at the early milky stage, playing a role in the down-regulation of GBSSI and SBE (starch branching enzyme) expression and the upregulation of SSS (soluble starch synthase) expression in developing grains under controllable growth conditions. Here, the decreased amylose content and longer amylose chains determined in GPC were possibly caused by inhibition of GBSSI expression and activity. GBSSI and SSSI are two major enzymes associated with grain quality and properties.43 Several studies have shown that SSSI accounts for about 60−70% of the total SSS activity, playing a distinct role in the synthesis of DP 8−12 chains and external segments of B chains.44 Thus, the up-regulation of SSS isoforms might be the main reason that rice starch from hot season tend to contain reduced numbers of short-chain and increased numbers of long-chain amylopectin. Crystalline Starch Structure. The X-ray diffraction (XRD) patterns of four rice starch samples were similar (Figure 7A), displaying a typical A-type diffraction pattern, with strong diffraction peaks at around 15° and 23° 2θ and an unresolved doublet at around 17° and 18° 2θ, which is in agreement with the XRD patterns of normal cereal starch.45 XRD also provides data on the relative amounts of crystallites and amorphous phases to help elucidate the crystal structures

from the two rice varieties showed a unimodal peak in size distribution, the average particle size of starch granules of Nanjing46 grown in 2013 (3.99 ± 0.98 μm) was smaller than that from 2014 (4.55 ± 0.78 μm). In contrast, no significant difference in average particle size was observed for Jia58. Chalky regions of rice grains tend to contain loosely packed, round starch granules with large air spaces.35 Thus, the significant difference in morphological structure and average particle size for Nanjing46 in different years might be due to the high PGWC in 2013. To further study the swelling characters of isolated starch, we investigated their swelling powers from 55 to 95 °C at 5 °C intervals. As shown in Figure 4E,F, the swelling power of each sample dramatically increased from 55 to 65 °C and from 85 to 95 °C. N-2013 [H] starch had much higher swelling power throughout the range of temperatures (55−75 °C) than N2014 [L] starch. A similar tendency was observed for Jia58 starch, with J-2013 [H] starch having a higher swelling power than J-2014 [L] starch. Prior to the disruption of swelling granules during RVA analysis, the viscosity is largely determined by the volume occupied by the starch granules.36 Therefore, the above results clearly show that starch granules from 2013 exhibited a higher swelling volume and swelling power during heating than those from 2014, which might result in the higher peak viscosity revealed by RVA. Fine Starch Structure. We analyzed the fine structure of starch from the above four samples. They were completely debranched and separated by GPC, and three well-resolved fractions were observed (Figure 5A,B). On the basis of other reports, we determined that the Ap1 and Ap2 fractions comprise amylopectin, and the Ap1 fraction usually contains low-molecular-weight molecules such as A and short B chains (A + B1 chains), and the Ap2 fraction is mainly composed of

Figure 5. Fine structure of debranched starch as determined by gel permeation chromatograms. Panels A and C represent the cultivar Nanjing46, and panels B and D represent the cultivar Jia58. Panels C and D indicate the amylose fractions from panels A and B, respectively. N-2013 [H], N-2014 [L], J-2013 [H], and J-2014 [L] represent samples from the cultivars Nanjing46 (indicated as N) and Jia58 (indicated as J) grown in 2013 with a hot season (high temperature) [H] and 2014 with a normal season (low temperature) [L], respectively. MW indicates the apparent molecular weight relative to the standards. 4053

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Journal of Agricultural and Food Chemistry Table 3. GPC, Crystallinities, and IR Ratio Parameters of Rice Starch cultivarsa Nanjing46-2013 [H] Nanjing46-2014 [L] Jia58-2013 [H] Jia58-2014 [L]

Ap1/Ap2 (area ratio) 2.91 3.12 2.56 2.82

± ± ± ±

0.01* 0.02** 0.07* 0.08**

,b

Am1/Am2 (area ratio) 1.13 0.95 1.15 0.91

± ± ± ±

0.03* 0.04** 0.08* 0.07**

crystallinity (%) 34.86 32.98 35.58 33.49

± ± ± ±

0.13* 0.05** 0.52* 0.68**

IR ratio of 1045/1022 (cm−1) 0.616 0.601 0.603 0.589

± ± ± ±

0.001* 0.001** 0.005* 0.013**

a

[H] and [L]: hot (high temperature) and normal (low temperature) season during the grain-filling stage, respectively. bValues with the same number of asterisks in a column of the same cultivar are not significantly different (p < 0.05).

Figure 6. Changes in the chain length distribution of amylopectin from isoamylase-debranched starch between the two growth season as determined by HPAEC. N-2013 [H], N-2014 [L], J-2013 [H], and J2014 [L] represent samples from cultivars Nanjing46 (indicated as N) and Jia58 (indicated as J) grown in 2013 with a hot season (high temperature) [H] and 2014 with a normal season (low temperature) [L], respectively.

of starch crystallites. The relative crystallinity calculated from XRD patterns showed significant differences between rice cultivars grown in different years (Table 3). Both cultivars had a significantly higher degree of crystallinity in 2013 than in 2014, which might be due to the differences in amylopectin structure and amylose content. Amylopectin is generally considered to be responsible for starch crystallinity, and amylose disrupts the crystalline packing of amylopectin.46 Therefore, the increase in intermediate amylopectin chains in both cultivars in 2013 might be the main reason for the increased crystallinity. In addition, rice grains harvested in 2013 contained lower amylose levels, which might also explain their higher crystallinity. The above results are consistent with previous studies of rice grown at high ripening temperatures.8 ATR−FTIR is thought to be sensitive to the short-range order, i.e., the double-helix content in starch, which has been used to study starch granule crystallinity and the amorphous regions near the granule surface.47 The FTIR intensity ratio of bands at 1022 and 1045 cm−1 expresses the amount of ordered starch to amorphous starch and has been linked to the characteristics of amorphous and crystalline structures in starch, respectively. The 1045/1022 cm−1 ratio from the deconvoluted FT-IR spectrum can therefore be used as a convenient index of FTIR compared to other measurements of starch conformation.47 The deconvoluted FTIR spectra of starch were similar among the four rice-starch samples (Figure 7B). However, on the basis of the calculation of relative intensities of FTIR bands at 1045 and 1022 cm−1 from the baseline to peak height, we determined that the starch from 2013 tended to have higher 1045/1022 cm−1 ratios, which is in agreement with the data

Figure 7. XRD (A) and ATR−FTIR (B) spectra of purified starch. N2013 [H], N-2014 [L], J-2013 [H], and J-2014 [L] represent samples from cultivars Nanjing46 (indicated as N) and Jia58 (indicated as J) grown in 2013 with a hot season (high temperature) [H] and 2014 with a normal season (low temperature) [L], respectively.

from XRD analysis. These results suggest that the increased double helical ordered structure in the external region of starch granules from 2013 were mainly caused by the increase in intermediate (DP 13−34) and long chains (DP 45−63) of amylopectin, as determined by HPAEC. Thermal Starch Properties. The gelatinization properties of rice are closely related to eating quality. Therefore, rice grown at high temperatures would require higher cooking temperatures.2,48 We analyzed the thermal properties of the four starch samples by DSC. The starch of both cultivars presented similar gelatinization curves in the same growing year (Figure 8A) and showed no significant difference in GT, as determined by the alkali spreading method. However, for both varieties, the thermal properties of starch differed between the two seasons. An increase in gelatinization properties, such as onset (To), peak (Tp), conclusion (Tc), and enthalpy (ΔHgel), was observed in 2013 compared to observations in 2014 (Table 4). GT is not significantly correlated with amylose content,45,49 but it is significantly negatively correlated with the amount of 4054

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crystallinity than those from 2014, which mainly depends on the fine structure of amylopectin and the amylose-toamylopectin ratio. Kong et al. showed that the gelatinization enthalpy of rice starch is negatively correlated with amylose content.53 Similar results were obtained in the present study, i.e., the gelatinization enthalpy of rice starch with relatively low amylose content increased. These findings are also consistent with those of previous studies suggesting that the DSC characteristics of rice are correlated with environmental temperatures.8,48 Retrogradation of the starch was also observed after the gelatinized samples were stored at 4 °C for 7 days (Figure 8B). Both samples exhibited similar DSC characters in retrogradation, and there was a slightly higher To and ΔHret for Nanjing46 grown in 2013 than in 2014 (Table 4). During retrogradation, amylose forms double-helical associations of 40−70 glucose units, whereas amylopectin crystallization occurs by the association of the outermost short branches.39 A higher proportion of long B1 chains and average chain length (CL) contribute to the higher levels of recrystallized domains in waxy starch.29,55 Thus, the different To and ΔHret in Nanjing46 during retrogradation might be due to the higher Ap1-to-Ap2 ratio. Over the years, wide-ranging studies on the impact of different environmental factors on rice starch structure and functional performance in controlled-environment experiments have been reported, whereas few studies have been performed under open-field conditions, especially in different years with extreme weather events. The present study shows that hot temperatures during the grain-filling stage can significantly affect rice ECQ. In addition, these hot conditions led to higher PGWC, reduced protein and amylose contents, and higher pasting properties. However, these results partially contradict the previous finding that rice grains with relatively low amylose contents and higher GC tend to have good ECQ in Asia.9 These seemingly contradictory results might be due to other important grain and starch properties: starch in rice grown at high temperatures in the current study had higher PGWC and protein content and fewer short amylopectin chains (DP 5−12) but more intermediate (DP 13−34) and long (DP 45−60) amylopectin chains than that under normal temperatures. Therefore, PGWC, protein content, and amylopectin and amylose fine structure had the greatest relative effects on ECQ in rice grown in the field.

Figure 8. Gelatinization (A) and retrogradation (B) of purified starch at 66.7% (w/v) water content as determined by differential scanning calorimetry. N-2013 [H], N-2014 [L], J-2013 [H], and J-2014 [L] represent samples from cultivars Nanjing46 (indicated as N) and Jia58 (indicated as J) grown in 2013 with a hot season (high temperature) [H] and 2014 with a normal season (low temperature) [L], respectively.

short branch-chain amylopectin.50,51 Starch with relatively high levels of long branch chains requires higher temperatures for complete dissociated.52 Thus, the higher of gelatinization properties of samples from 2013 observed in this study might be due to reduced levels of short branch-chain amylopectin and higher levels of longer branch-chain amylopectin in starch, which is consistent with previous studies of diverse rice cultivars.53 Cooke and Gidley showed that the endothermic peak in DSC reflects the loss of double helices in amylopectin and that the total energy reflects the crystalline structure or molecular order.54 The higher enthalpy of samples from 2013 implies that they require more energy to melt starch

Table 4. Differential Scanning Calorimetry Characteristics of the Rice Flour cultivarsa gelatinization

retrogradation

Nanjing46-2013 Nanjing46-2014 Jia58-2013 [H] Jia58-2014 [L] Nanjing46-2013 Nanjing46-2014 Jia58-2013 [H] Jia58-2014 [L]

To (°C)b [H] [L]

[H] [L]

61.30 60.30 61.40 60.55 45.30 46.10 47.10 47.25

± ± ± ± ± ± ± ±

Tp (°C)b

0.14*,c 0.14** 0.14* 0.07** 0.14* 0.28** 0.07* 0.07*

67.45 65.45 67.25 65.40 54.01 55.00 54.70 54.65

± ± ± ± ± ± ± ±

0.50* 0.35** 0.07* 0.28** 0.14* 0.28* 0.28* 0.07*

ΔH (J G−1)b

Tc (°C)b 75.00 73.45 73.85 72.70 61.50 61.50 61.30 61.25

± ± ± ± ± ± ± ±

0.28* 0.21** 0.07* 0.25** 0.42* 0.28* 0.28* 0.35*

13.19 12.59 13.24 12.78 4.06 3.59 3.51 3.51

± ± ± ± ± ± ± ±

0.02* 0.01** 0.06* 0.26** 0.06* 0.17** 0.27* 0.09*

a

[H] and [L]: hot (high temperature) and normal (low temperature) season during the grain-filling stage, respectively. bTo, onset temperature; Tp, peak temperature; Tc, conclusion temperature; ΔH, enthalpy of gelatinization. cValues with the same number of asterisks in a column of the same cultivar are not significantly different (p < 0.05). 4055

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(9) Calingacion, M.; Laborte, A.; Nelson, A.; Resurreccion, A.; Concepcion, J. C.; Daygon, V. D.; Mumm, R.; Reinke, R.; Dipti, S.; Bassinello, P. Z.; Manful, J.; Sophany, S.; Lara, K. C.; Bao, J.; Xie, L.; Loaiza, K.; El-hissewy, A.; Gayin, J.; Sharma, N.; Rajeswari, S.; Manonmani, S.; Rani, N. S.; Kota, S.; Indrasari, S. D.; Habibi, F.; Hosseini, M.; Tavasoli, F.; Suzuki, K.; Umemoto, T.; Boualaphanh, C.; Lee, H. H.; Hung, Y. P.; Ramli, A.; Aung, P. P.; Ahmad, R.; Wattoo, J. I.; Bandonill, E.; Romero, M.; Brites, C. M.; Hafeel, R.; Lur, H. S.; Cheaupun, K.; Jongdee, S.; Blanco, P.; Bryant, R.; Thi Lang, N.; Hall, R. D.; Fitzgerald, M. Diversity of Global Rice Markets and the Science Required for Consumer-Targeted Rice Breeding. PLoS One 2014, 9, e85106. (10) Tian, Z. X.; Qian, Q.; Liu, Q. Q.; Yan, M. X.; Liu, X. F.; Yan, C. J.; Liu, G. F.; Gao, Z. Y.; Tang, S. Z.; Zeng, D. L.; Wang, Y. H.; Yu, J. M.; Gu, M. H.; Li, J. Y. Allelic diversities in rice starch biosynthesis lead to a diverse array of rice eating and cooking qualities. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 21760−21765. (11) Hizukuri, S. Polymodal distribution of the chain lengths of amylopectins, and its significance. Carbohydr. Res. 1986, 147, 342− 347. (12) Tester, R. F.; Karkalas, J.; Qi, X. Starch-composition, fine structure and architecture. J. Cereal Sci. 2004, 39, 151−165. (13) Blazek, J.; Gilbert, E. P. Application of small-angle X-ray and neutron scattering techniques to the characterisation of starch structure: A review. Carbohydr. Polym. 2011, 85, 281−293. (14) Ratnayake, W. S.; Jackson, D. S. A new insight into the gelatinization process of native starches. Carbohydr. Polym. 2007, 67, 511−529. (15) Lin, L. S.; Cai, C. H.; Gilbert, R. G.; Li, E. P.; Wang, J.; Wei, C. X. Relationships between amylopectin molecular structures and functional properties of different-sized fractions of normal and highamylose maize starches. Food Hydrocolloids 2016, 52, 359−368. (16) Yamakawa, H.; Hirose, T.; Kuroda, M.; Yamaguchi, T. Comprehensive expression profiling of rice grain filling-related genes under high temperature using DNA microarray. Plant Physiol. 2007, 144, 258−277. (17) Liu, X.; Wan, X.; Ma, X.; Wan, J.; Gulick, P. Dissecting the genetic basis for the effect of rice chalkiness, amylose content, protein content, and rapid viscosity analyzer profile characteristics on the eating quality of cooked rice using the chromosome segment substitution line population across eight environments. Genome 2011, 54, 64−80. (18) Dang, J. M. C.; Copeland, L. Genotype and environmental influences on pasting properties of rice flour. Cereal Chem. 2004, 81, 486−489. (19) Lyman, N. B.; Jagadish, K. S. V.; Nalley, L. L.; Dixon, B. L.; Siebenmorgen, T. Neglecting rice milling yield and quality underestimates economic losses from high-temperature stress. PLoS One 2013, 8, e72157. (20) Battisti, D. S.; Naylor, R. L. Historical warnings of future food insecurity with unprecedented seasonal heat. Science 2009, 323, 240− 244. (21) Lobell, D. B.; Schlenker, W.; Costa-Roberts, J. Climate trends and global crop production since 1980. Science 2011, 333, 616−620. (22) Patindol, J. A.; Siebenmorgen, T. J.; Wang, Y. J. Impact of environmental factors on rice starch structure: A review. Stärke 2015, 67, 42−54. (23) Aboubacar, A.; Moldenhauer, K. A. K.; McClung, A. M.; Beighley, D. H.; Hamaker, B. R. Effect of growth location in the United States on amylose content, amylopectin fine structure, and thermal properties of starches of long grain rice cultivars. Cereal Chem. 2006, 83, 93−98. (24) Zhang, C. Q.; Zhu, L. J.; Shao, K.; Gu, M. H.; Liu, Q. Q. Toward underlying reasons for rice starches having low viscosity and high amylose: physiochemical and structural characteristics. J. Sci. Food Agric. 2013, 93, 1543−1551. (25) Sandhu, K. S.; Singh, N. Some properties of corn starches II: physicochemical, gelatinization, retrogradation, pasting and gel textural properties. Food Chem. 2007, 101, 1499−1507.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b00083. A table showing the pasting properties of flour and starch from the two rice cultivars grown in 2013 and 2014. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 514 8797 9242; e-mail: [email protected]. Author Contributions ∥

C.Z. and L.Z. contributed equally to this work.

Funding

This study was financially supported by the National Natural Science Foundation (31401354 and 31561143008), the Ministry of Agriculture (2014ZX08009-024B), the Jiangsu Natural Science Foundation (BK20140484), the Ministry of Education (20133250120001), the JAAS (ZX(15)4003), and the Jiangsu Department of Education (PAPD, 201411117015Z, and KYLX15_1372). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED AAC, apparent amylose content; ATR−FTIR, attenuated total reflectance−Fourier transform infrared; BDV, breakdown viscosity; CPV, cool paste viscosity; DP, degree of polymerization; DSC, differential scanning calorimeter; ECQ, eating and cooking quality; GBSSI, granule-bound starch synthase I; GC, gel consistency; GPC, gel-permeation chromatography; GT, gelatinization temperature; HPAEC, high-performance anion-exchange chromatography; Ptemp, pasting temperature; PC, protein content; PGWC, percentage of grains with chalkiness; RVA, Rapid Visco Analyzer; To, onset temperature of gelatinization; ΔH, enthalpy of gelatinization; SP, swelling power; TSC, total starch content; XRD, X-ray powder diffraction

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