Effects of Late-Stage Nitrogen Fertilizer Application on the Starch Structure and Cooking Quality of Rice
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XianMei Cao, HuiYan Sun, ChunGe Wang, XiaoJia Ren, HongFei Liu, ZuJian Zhang*
Jiangsu Key Laboratory of Crop Genetics and Physiology/Key Laboratory of Plant Functional Genomics, Ministry of Education of China, Agricultural College of Yangzhou University, 225009, China
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Abstract BACKGROUND: With the rapid development of modern agriculture, high-quality rice production and consumption has become the current urgent demand for the development of rice production. RESULT: In this paper, the effects of late-stage nitrogen fertilizer application on rice quality were studied under the same genetic background, Wx near-isogenic lines were used as test materials to study the starch composition, amylopectin structure, and cooking quality of rice. Results showed that rice amylose content and gel consistency This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.8723
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significantly differed when different Wx genes were tranformed into waxy rice, the law of apparent amylose content in rice is Wxa>Wxin>Wxb>wx at the same nitrogen
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level, while the trend of gel consistency was opposite to that of apparent amylose content, presenting obvious characteristics of Indica and Japonica varieties. As the amount of fertilizer application increased, apparent amylose content increased, gel consistency decreased, breakdown and peak viscosities dropped, and setback viscosity and peak time increased. Moreover, the cooking quality of rice significantly decreased with the use of nitrogen fertilizer, especially under low-level nitrogen fertilizer application. Amylopectin structure varied significantly in different genotypes of the Wx gene, and the degree of branching was as follows: wx>Wxb>Wxin>Wxa. This result indicated that the closer to Indica rice, the less short chains of amylopectin. Starch crystallinity and swelling potential were negatively correlated with amylose content but significantly positively correlated with amylopectin branching degree, decreasing with the increase of late-stage nitrogen fertilization. CONCLUSION: the late-stage nitrogen fertilization reduced the cooking quality of rice by increasing amylose content, reducing amylopectin branching degree, which decreased starch crystallinity, and aggravated pasting properties. Obviously, controlling late nitrogen application is essential to optimize rice quality. Key words: rice, Wx gene, nitrogen fertilizer, cooking quality, amylose, amylopectin 1. Introduction Starch weight accounts for 85%–90% of polished rice, which is mainly composed of amylose and amylopectin at 0%–30% and 70%–95% of rice starch, This article is protected by copyright. All rights reserved.
respectively.1-3 Amylose is a linear glucan chain with alpha-D-glucose units connected by the alpha-1,4-glycosidic bond, unbranched or rarely branched. The chain length is
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in the range of 1000–6000 DP. The amylopectin molecules in the branch are connected with alpha-1,4-glycosidic bond; the branches are connected with alpha-1,6-glycosidic bond, and the average branch length is 20–25 DP.4 Starch is lined with amylose and amylopectin molecules, with an alternating layer of crystalline and amorphous areas. The crystalline area constitutes a fixed layer of starch granules, and the amorphous area constitutes a sparse layer of starch granules. These areas are lined alternately.5,6 Hizukuri pointed out that amylopectin branches are not random but are clustered. Starch properties are determined not only by the amylose content but also by the distribution pattern of various branches of the cluster structure.7 The fine structure of amylopectin mainly includes the average polymerization degree, branching degree, average chain length, and chain length distribution.8,9 The average polymerization degree is 8200–12800. In general, the average polymerization degree in Indica rice is lower than that in Japonica rice. Higher branching degree of starch facilitates easy gelatinization of starch.10 Nakamura et al. showed that the structure of rice cultivars can be classified according to the different chain length distributions of amylopectin: L and S types. The L type possesses less short-length chain (DP≤11) and more medium-long length chain （DP=12–24）than the S type. Most Indica varieties belong to the L type.11 The amylose content in rice is mainly regulated by the Wx gene located on the short arm of the sixth chromosome.12 Different rice varieties including mainstream Indica, Japonica, tropical Japonica, and waxy varieties contain different
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Wx alleles. The Wx gene includes Wxa, Wxb, Wxin, and wx. Furthermore, nonwaxy gene Wx displays dominant expression on the waxy gene wx and evidently exerts a
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dose effect.13,14 Zhou et al. showed a similar rapid visco analyzer (RVA) profile for the five alleles of Wxa, Wxin, Wxb, Wxmq, and wx with a significant difference in amylose content. Nevertheless, the RVA value shows a significant difference.15 Umemoto et al. constructed the near-isogenic line using the genetic background of Nipponbare; the rice amylose content in the Wxa genotype is significantly higher than that in the parents, and the RVA spectrum characteristic values of peak viscosity (PKV) and breakdown viscosity (BDV) are significantly lower than those in the parents.16 In addition, amylopectin structure also exerts a remarkable effect on the cooking and eating qualities of rice. The amylose content of some high-quality Japonica Rice shows no significant difference with that of Koshihikari, but the ratio of short/long chain in amylopectin is higher in the former than in the latter.17 Cai et al. showed that the ratio of short chain part (Fr III) of branched chain starch is significantly positively correlated with PKV and BDV, but the long chain part (Fr (Ⅰ+Ⅱ)) displays an inverse correlation.18 Cheetham and Tao showed a strong correlation between the short/long chain ratio in amylopectin and starch crystallinity, i.e., higher ratio corresponds to higher crystallinity.19 However, the indirect effect of the Wx gene on starch structure is rarely reported. Nitrogen is an important and non-negligible environmental factor affecting rice quality. Although the effects of nitrogen fertilizer on the properties of rice starch were verified in numerous studies, combining the trends and observation is difficult.20-22
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Many studies suggested that nitrogen fertilizer is not contributing to rice quality improvement, but many research results showed that appropriate nitrogen can
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maintain and improve rice quality. Gu et al. suggested that the PKV, hot paste viscosity (HPV), cool paste viscosity (CPV), and BDV of starch decrease with increasing nitrogen fertilizer level; additionally, the eating quality is aggravated.23 Singh et al. reported that starch from rice grown with nitrogen application contains low amylose level.24 Duan concluded that middle or high nitrogen fertilizer level increases the short chain proportion of amylopectin and decreases the long chain proportion under water-saving conditions, thereby improving the quality of rice.25 The effect mechanism of nitrogen fertilizer application on rice synthesis is poorly understood. This mechanism is related to the test materials with complex genetic background. Conclusion vary due to the differences in varieties and environment used among studies. The present research used different Wx near-isogenic lines and prepared different fertilizer treatments to determine the effects of the Wx gene on rice quality and response characteristics of nitrogen under consistent genetic background. This study widens understanding of rice quality and provides the necessary theoretical basis for high-quality cultivation.
2. Materials and Methods 2.1 Test materials and growth conditions The experiment was carried out in 2015 and 2016 in the experimental field of the Agricultural College of Yangzhou University (32° 30＇N, 119° 30＇E). The condition
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of the test materials is shown in Table 1. Wx near-isogenic lines were used. Yangfunuo 4 was the recipient parents carrying four Indica rice cultivars (Minghui 63, Teqing,
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IR36, and IR64) with different Wx genes as donor parents; these lines were crossed and returned. The near-isogenic lines carrying different Wx genes were screened with the genetic background of Yannuo 4 through molecular marker-assisted selection. The soil in experimental site was a sandy loam with 23.2 g kg−1 organic matter, 98.2 mg kg−1 alkali-hydrolyzable N, 32.7 mg kg−1 Olsen-P, and 87.5 mg kg−1 exchangeable K. The previous crop was wheat.
The seeds were first sown in the paddy field on May
12. The seedlings were subsequently transplanted to the field on June 12 at a hill spacing of 20 × 18 cm with two seedlings per hill and harvested on October 6. Plot dimensions were 5 m × 3 m. Nitrogen treatment (N1: 0 kg N ha–1, N2: 60 kg N ha–1, N3: 120 kg N ha–1) was carried out seven days before heading, and each treatment was repeated three times. Basic fertilizer consisting of compound fertilizer (750 kg ha–1) and urea (150 kg ha–1) was applied uniformly. Tillering fertilizer was also evenly applied using urea (150 kg ha–1). The harvest was threshed and naturally dried. The seeds were stored for 3 months for rice quality determination. Rice powder was sieved over 100 mesh.
2.2 Gel consistency and amylose content analysis Gel consistency was determined in accordance with GB1350-1999. Amylose content was determined in accordance with the Ministry of Agriculture ministerial standard NY147-88.26
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2.3 Determination of starch gelatinization properties The viscosity tester RVA (Newport Scientific Instruments, Australia) was used in
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accordance with the American Society for Cereal Chemistry operating procedures (1995 61-02). The RVA spectrum characteristic value was mainly expressed by the peak viscosity (PKV), hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BDV, PKV–HPV), setback viscosity (SBV, CPV–PKV), consistence viscosity (CSV, CPV–HPV), peak time（PeT）, and pasting temperature (PaT).
2.4 Extraction of starch Approximately 10 g of rice flour was placed in a 50 mL centrifuge tube. Subsequently, NaOH solution (10-5 mol/L) was added to a 35 mL centrifuge tube; each tube contained 50 g kg-1 alkaline protease. After magnetic stirring under 42 °C constant temperature for 24 h, the homogenate was filtered through a 200 mesh sieve, and the filtrate was centrifuged at 4000 r min–1 for 20 min. The supernatant was discarded, and the yellow portion of the starch surface was removed. After centrifugation with deionized water, the extracts were centrifuged at 4000 r min-1 for 20 min. The above operation was repeated 3~5 times to remove ions and impurities. Afterward, 95% ethanol, chloroform/methanol mixture (V / V = 1: 1), and methanol/acetone mixture (V / V = 1: 1) were used to wash the extracts for 2~3 times and remove the fat. Baked until half dry, stirred with glass rods, starch was obtained by drying and screening over a 200-mesh sieve, then placed in a 4 °C refrigerator and
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sealed for storage.
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2.5 Determination of the branching degree of amylopectin For starch debranching treatment, 5 mg of starch was placed in a 2 mL centrifuge tube. Subsequently, 1.8 mL of DMSO/NaNO3 (0.5% W/W) was added. The mixture was placed at a constant temperature of 85 °C and dissolved at 33×g overnight. Centrifugation was performed at 1467×g for 10 min, and 1.5 mL of supernatant was carefully aspirated and placed in a 10 mL centrifuge tube. Afterward, anhydrous ethanol (four times of the volume) was added. The solution was mixed and centrifuged at 3724×g for 10 min. The supernatant was discarded, and the procedures were repeated three times. Approximately 1.0 mL of ultrapure water was added, and the solution was boiled for 1 h. Afterward, 0.9 mL of solution was obtained and placed in a 2 mL centrifuge tube; 0.1 mL of sodium acetate buffer (0.1 mol/L), 5 µL of sodium azide solution (0.04 g/mL), and 3.5 µL of amylase solution were also added. The resulting solution was vortexed and mixed at 37 °C overnight. About 180 µL of NaOH (0.1 mol/L) was added to adjust the pH of the solution to 7. For amylopectin GPC analysis, the off-branch of the sample was frozen with liquid nitrogen and placed in a freeze dryer. Approximately 1.8 mL of DMSO was added, and the temperature was set to 85 °C. The mixture was dissolved at 33×g overnight. After filtering at 0.45 µm, condensates were used for GPC (LP-GPC220, British PL). Three starch columns, namely, PL1110-6100, PL1110-6300, and PL1110-6525 were used for analysis.
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2.6 Starch grain type and crystallinity determination
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The starch was immersed in a sealed container containing a saturated aqueous solution of sodium chloride for one week and then subjected to an X-ray diffraction analyzer (D8 type multi-X diffractometer, Bruker AXS, Germany) under the following conditions: current, 40 mA; voltage, 40 kV; diffraction angle, 3°–40°; scanning speed, 0.3 s; and step size, 0.02°. The X-ray diffraction spectrum of the sample starch was measured twice a day. Origin75 software was used for mapping.
2.7 Determination of swelling potential and solubility A mixture of starch sample (30 mg) and ultrapure water (1 mL) in a 2 mL Ep tube (A) was place in shaking water bath at 90 °C for 1h. The mixture was subsequently centrifuged (4000×g) for 10min. The supernatant was placed in another 2 mL EP tube (B), and dried at 60 °C. The colloid adhering to the A pipe wall was weighed to obtain its water swelling weight. Solubility (%) = dissolved sample weight/ sample weight×100; Swelling power (g/g) = weight after water swelling/(sample weight – weight of dissolved sample).
2.8 Data processing Microsoft Excel software was used to organize data, and SPSS16.0 software was applied for statistical treatment.
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3. Results
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3.1 Effects of late-stage fertilization on the cooking quality of different Wx genotypes Table 2 shows that the apparent amylose content of rice is Y3, Y5 (Wxa)> Y7 (Wxin)> Y1 (Wxb)> Y9 (wx) at the same nitrogen level. This result showed significant rice starch formation among the Indica, Japonica, and waxy varieties during the two years. With the increase in late-stage fertilization level, the apparent amylose content increased, and the trend of gel consistency was opposite to that of apparent amylose content. The decreasing trend of gel consistency was more evident than that of amylose content. Reaction differences were observed among genotypes. The gel consistency of Y3, Y5 (Wxa genotype), and Y7 (Wxin genotype) rice varieties largely decreased 33.7 mm and 32.0 mm with the increase in fertilization levels in 2016. Such decline showed the sensitivity of rice gel consistency to late nitrogen fertilizer, and Y9 (wx genotype) rice remained essentially unchanged. Thus, in terms of cooking traits, Y3 and Y5 (Wxa genotype) obtained from Indica were relatively sensitive to late-stage fertilization, Y1 (Wxb genotype) was relatively insensitive, and Y9 (wx genotype) was insensitive. In addition, the amylose content showed significant difference in interannual, especially Y3, Y5 (Wxa genotype), and Y7 (Wxin genotype); this result indicated that these two genotypes were easily affected by climate factors.
Table 3 shows the response of the RVA profile of different Wx genotypes to
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late-stage fertilization. Among the different Wx genotypes, the BDV and PKV of Y1 (Wxb genotype) were the highest, and those of Y3 and Y5 (Wxa genotype) were
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relatively low. The CSV, SBV, and PeT of Y3 and Y5 (Wxa genotype) were relatively high, and those of Y9 (wx genotype) were the lowest; Y9 (wx gene type) and Y1 (Wxb genotype) showed high PaT of rice starch. With the increase in nitrogen fertilizer level in the late stage, the BDV and PKV were decreased, and the SBV and PeT of each material were increased, leading to cooking quality deterioration. The CSV and PaT were relatively stable. Considering the nitrogen fertilizer response differences among the genotypes, Y1 (Wxb genotype) and Y9 (wx genotype) were relatively insensitive, and Y3, Y5 (Wxa genotype), and Y7 (Wxin genotype) changed considerably, especially in 2015. Compared with varying from N1 to N2 levels, the amplitude of decrease in BDV and increase in SBV and PeT both were greater than those varied from N2 to N3 levels in all varieties. These results showed that starch cooking quality was sensitive to low nitrogen fertilizer.
3.2 Structural characteristics of amylopectin in rice with different Wx genotypes Figure 1 shows the molecular genotype of rice starch measured in Wx genotype distribution after isoamylase debranching. Except for waxy gene, the four genotypes exhibited typical three-peak distribution, but the peak level was significantly different. The peak area of Peak1 in the graph indicated the percentage of amylopectin short chain, the peak area of Peak2 indicated the percentage of long chain in amylopectin, and the peak area of Peak3 indicated amylose content. The ratio of Peak1/Peak2 is
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commonly regarded as the branching degree of amylopectin; higher ratio corresponds to higher amylopectin branching degree. Graphical data processing results are shown
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in Table 4.
Table 4 shows that Y9 (wx genotype) is a glutinous rice; it contains no amylose. The amylose content of Y3 and Y5 (Wxa genotype) rice was the highest. Distribution analysis on the amylopectin chain showed that short chain was proportional to the size of the trend of Y9 (wx)>Y1 (Wxb)>Y7 (Wxin)>Y3 and Y5 (Wxa). This result suggests that closeness to Indica corresponds to less short-chain proportion. With the increase in late-stage N level, except in Y1 (Wxb genotype), the percentage of short chain accounted in all genotypes exhibited a downward trend, whereas that of long chain was relatively the contrary; Y1 (Wxb genotype) also showed an evident increasing trend. The ratio of the length of the short chain to the middle-long chain represents amylopectin branching degree, and the branching degree decreased with increasing late-stage fertilization level. Variance analysis showed a significant difference in the short chain and branching degree of amylopectin under different fertilizer treatments, and their effects on amylose content exerted significant differences in materials. Therefore, late-stage fertilizer application significantly influenced the starch structure. A significant difference existed between the materials and fertilizers in the short chain of amylopectin, especially in the short-chain proportion of Y1 (Wxb genotype) responding to the fertilizer application, which were significantly different with other materials. The late-stage nitrogen fertilizer application resulted in decreased short
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chain proportion of Indica Wxa material and increased long chain proportion of
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Japonica Wxb material, which consequently reduced the starch branching degree.
Analysis on the amylose content, amylopectin branching degree and starch gelatinization characteristics (RVA profile) in Table 5 showed that the amylose content correlated negatively and negatively significantly with BDV and PaT (correlation coefficients: –0.624* and –0.662**), respectively. On the contrary, amylose content showed significantly positive correlation with the CPV, SBV, CSV, and PeT (correlation coefficients: 0.890**, 0.929**, 0.980**, and 0.942**, respectively). A significant positive correlation also existed between the branching degree of amylopectin and the PKV, BDV, and PaT; their correlation coefficients were 0.668**, 0.856**, and 0.598*, respectively. By contrast, significantly negative correlation existed between the amylopectin branching degree and the CPV, SBV, CSV, and PeT (correlation coefficients: –0.626*, –0.909**, –0.753**, and –0.743**, respectively). The amylose content or starch branching degree exhibited no significant correlation with HPV.
3.3 Crystallinity and swelling characteristics of rice starch with different Wx genotypes Figure 2 shows the X-ray diffraction patterns of the different genotypes of rice starch. The diffraction pattern of rice starch was all type A, and the 2θ angle was mainly at 15°, 17°, 18°, and 23°. This result indicated that the fertilization level
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exerted no influence on the crystal-type rice starch, but some differences existed in the peak intensity. The relative crystallinity in Table 6 showed that the relative
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crystallinity trend was Y9 (wx)> Y1 (Wxb)> Y7 (Wxin)> Y3 and Y5 (Wxa) at the same fertilizer level. Except for Y9 (wx), the relative crystallinity decreased with increasing nitrogen fertilizer level. Additionally, the decreasing range of Y7 (Wxin genotype) and Y3 and Y5 (Wxa genotype) rice was larger. The crystal formation of Wxin and Wxa rice starch was relatively sensitive to late-stage fertilizers.
The swelling potential and solubility of the rice starch for each genotype material are shown in Table 7. The water-soluble starch displayed advantages, such as water solubility, gelation, water retention, and swelling; these advantages were related to the cooking and eating quality of rice. The swelling power trend was Y9 (wx)> Y1 (Wxb)> Y7 (Wxin)> Y5 and Y3 (Wxa) at the same fertilizer level. Solubility showed a large value at Y9 (wx genotype), and the trend of others was opposite to that of starch swelling power. Swelling power decreased with increasing nitrogen fertilizer level. Y9 (wx genotype) rice decreased by 5.55%, and Y1 (Wxb genotype) was stable. Solubility increased with the application of nitrogen fertilizer. The increase of Y9 (wx genotype) was the highest at approximately 15.35%. The starch properties of Japonica materials were relatively stable in response to fertilizers.
Analysis results on the relationship of starch crystallinity and swelling properties or starch structure are shown in Table 8. The relative crystallinity and swelling power
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of starch showed a significant negative correlation with amylose content but a significant positive correlation with amylopectin branching degree. In terms of starch
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solubility, a significant positive correlation was found between solubility and amylose content, except for Y9 (wx genotype) rice, which showed a directly opposite trend to amylopectin branching degree.
3. Discussion 4.1 Performance characteristics of rice cooking quality in different Wx genotypes Amylose, PaT, and gel consistency are the main rice cooking quality evaluation index, and amylose content is of the most critical determinant.27-29 The Wx gene encodes the granule bound starch synthase in grains, which is responsible for the synthesis of amylose and the key gene to determine the amylose content of rice. The different Wx genotypes determine different rice cultivars. The different expression levels of the Wx gene in varieties cause the different amylose contents in the endosperm. The Wxa, Wxb, Wxin, and wx genotypes tested in this experiment are representatives of different rice genotypes. The different quality characteristics represent the performance of Indica, Japonica, tropical Japonica and glutinous rice types; these types were tested for several years, and they showed remarkably consistent performance.30,31 The change in amylose content caused adapted gel consistency and gelatinization temperature (Tables 2 and 3). In the RVA spectrum of visible characteristics, the RVA characteristics of the five materials also indicated the significant characteristics of Indica and Japonica. The BDV and PKV were the highest
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in the Wxb genotype, whereas relatively low in the Wxa genotype. The CSV, SBV, and PeT in the Wxa genotype were relatively high, whereas the lowest in wx. The test
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materials are of the same genetic background but with different near-isogenic lines of the Wx gene. Thus, the amylose content difference due to the Wx gene is the main reason for the different characteristics of the rice RVA. Jang et al. showed that Indica rice contains higher amylose, protein, and starch particles than Japonica rice, thereby showing higher gelatinization temperature at low viscosity. Additionally, amylose content and gelatinization temperature were positively correlated, but the PKV and BDV were significantly negatively correlated.32 However, in this study, the gelatinization temperature and amylose content were negatively related. Furthermore, rice quality is a quantitative trait that resulted from gene–environment interactions. Many previous studies using different varieties as test materials cannot exclude the influence of the genetic background, which may also be an important factor in varying research conclusions. Consequently, the quality of rice cultivation and regulation cannot be easily obtained. Given a new basis by using near-isogenic lines of molecular marker-assisted selection method to study the physiology and cultivation regulation, this method can not only analyze the biological effect of one specific gene, but also analyze the environmental effects without the interference of genetic background in variety, thereby obtaining a reliable conclusion. Correlation analysis determining the effect of the four Wx genotypes of five near-isogenic lines on the cooking quality of rice verified the significance of screening existing knowledge. 4.2 Chain length distribution of amylopectin in different Wx genotypes and their
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effects The Wx gene is the major gene that directly determines the amylose content.
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Different Wx alleles determine the difference between rice varieties. The results also showed that the Wx genotype of the different rice amylopectin chain length distribution exhibited differences. The short chain was proportional to the size with the following trend: wx>Wxb>Wxin>Wxa. The middle-long chains showed a trend opposite to that of the short chain. The trend of starch branching performance and short chain proportion was consistent; the trend changed from Indica to Japonica under the same genetic background, the amylose content declined, the amylopectin branching increased, and short chain increased. The Wx gene and amylopectin showed no evident change because no correlation existed between the amylopectin synthesis and biological function of the Wx gene. Therefore, the influence of the Wx gene on starch can be indirect. Starch synthesis in grains is an organic process. Each related gene and the corresponding enzyme form the whole network of starch synthesis and cause phase differences. For example, the Wx gene increases amylose content. Amylopectin adapts to the inevitable reduction in the formation of amylopectin, which also exerts a corresponding change degree and forms different amylopectin structures. In order to evaluate the indirect effect, only when Wx gene expression was the exclusive variable under premise of the same genetic background, can correct result be obtained. In addition, Wx gene synthesis effect on grain starch was mainly reflected in the amylose content and starch complex effect. The impact pathway should also be considered to further understand the mechanism of rice quality
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analysis. Starch is a natural crystalline polymer. Starch grain structure contains the crystal
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and amorphous phases of the two major components. Amylopectin mainly forms a crystalline zone, and amylose mainly forms an amorphous zone. The external environment cannot change the crystal type of starch, but it can change the crystallinity of starch.33-35 Results showed that the grain types of the Wx genotype were the same. Nevertheless, the relative crystallinity was wx> Wxb> Wxin> Wxa and that of Japonica rice was higher than that of Indica rice. Late-stage nitrogen application exerted no influence on the type of rice starch crystals, but it reduced the relative crystallinity. The relative crystallinity of rice starch was significantly negatively correlated with amylose content, while was significantly positively correlated with the branching degree of amylopectin. Hence, under uniform genetic background, crystallinity changes may be attributed to differences in starch composition and chain length structure and result in corresponding changes in starch swelling and solubility. Cai reported that the solubility of rice starch positively correlates with amylose content but negatively correlates with amylopectin short chain; rice starch swelling potential positively correlates with amylopectin and short chain but negatively correlates with amylose content.36 It is consistent with the results of this study. Furthermore, an inevitable correlation exists between rice starch crystallization characteristic and cooking quality. 4.3 Effects of nitrogen levels on the grain filling stage of starch structure and cooking and eating quality of rice
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The important role of nitrogen fertilizer in rice quality cannot be ignored. However, many conclusions regarding the effect of nitrogen fertilizer on rice quality
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are not consistent, which leads to the evaluation of the effect of nitrogen fertilizer on rice quality. In this study, we used the near-isogenic lines of the rice Wx gene with consistent genetic background. The different nitrogen fertilizer levels during grain filling were established through late-stage nitrogen fertilizer treatments. The responses of different Wx genes to nitrogen fertilizer level were also evaluated. Test results showed that the responses of wx, Wxb, Wxin, and Wxa to nitrogen fertilizer were mainly the same: with the increasing fertilization level, the apparent amylose content increased, the amylopectin branching ratio decreased, the crystallinity decreased, and the gel consistency decreased significantly. Consequently, the cooking quality of rice was sensitive to late-stage fertilizer application. RVA results also showed the same trend. In addition, low-level nitrogen fertilizer application can cause significant deterioration of the relevant gelatinization characteristics, which showed the sensitivity of rice quality to low-level nitrogen fertilizer. The negative effect of late nitrogen fertilizer application on rice quality was also significant. Inducing difference in starch synthesis by altering the Wx gene expression can affect rice cooking quality. The response intensity of the different Wx genotypes to nitrogen levels also exhibited differences. The Wxb and wx genotypes were relatively insensitive, and the Wxa and Wxin genotypes changed remarkably. The results showed that Indica rice and middle type rice were sensitive to nitrogen level, and Japonica rice was relatively stable. Therefore, the effect of N fertilization on rice quality differed among different rice
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varieties. Cultivation measures should also be adjusted accordingly. To date, nitrogen management has become a key cultivation measure in rice production to ensure high
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yield. However, ensuring high yield and quality is difficult during rice cultivation. China’s current production level is the world’s highest under the condition of the world’s highest level of fertilization. Balancing high quality and yield is a considerable challenge. The current results suggest that applying a high level of nitrogen fertilizer in the late stage decreases rice quality. Therefore, the late-stage application of nitrogen fertilizer should be controlled, and the high yield and high-quality cultivation technology system should be considered. The main technical approaches include the growth promotion during early stage, establishment of high-yield structure, and control of late-stage nitrogen fertilizer application to ensure high quality and strong growth. In addition, on the basis of controlling the application of nitrogen fertilizer in the late stage, appropriate water management measures, such as dry and wet irrigation are used to increase the root vigor and improve the growth activity of the rice filling and obtain high yield and quality. The effect of nitrogen application may also be reflected in the improved rice protein content and decreased eating quality. Hence, the effect of starch and protein syntheses on rice quality should be further investigated to determine the mechanism affecting rice quality.
5. conclusion Insertion of various Wx genes in waxy rice under the same genetic background resulted in significant differences in the amylose content and gel consistency of rice
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with different Wx genes, thereby showing evident characteristics of Indica and Japonica varieties. Moreover, amylopectin structure showed significant differences.
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The branching degree in different genotypes was wx>Wxb>Wxin>Wxa. This result showed that closeness to Indica resulted in less short chain proportion. Starch crystallinity and swelling power showed a corresponding change. Late-stage nitrogen fertilization increased amylose content and reduced significant amylopectin branching crystallinity, and gel consistency. Among the RVA characteristic spectrum, the BDV and PKV decreased, the SBV and PeT increased significantly, and the cooking quality significantly deteriorated. The Wxa and Wxin genotypes were more sensitive to late-stage fertilizer application than the Wxb and wx genotypes. These results suggest that the nitrogen fertilizer application exerts evident negative effect on the cooking quality of rice. Therefore, controlling the late-stage nitrogen application should be further studied.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (31371564)
References 1 Varatharajan V, Eric B, Structure-function relationships of starch components. Starch/Stärke 67: 55–68 (2015) 2 Kong XL, Chen YL, Zhu P, Sui ZQ, Corke H, Bao JS, Relationships among Genetic, Structural, and Functional Properties of Rice Starch. J. Agric. Food Chem 63:
This article is protected by copyright. All rights reserved.
6241−6248 (2015) 3 Patindol JA, Siebenmorgen TJ, Wang Y, Impact of environmental factors on rice
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starch structure: A review. Starch/Stärke 67: 42–54 (2015) 4 Wang K, Hasjim J, Wu AC, Li E, Henry RJ, Gilbert RG, Roles of GBSSI and SSIIa in determining amylose fine structure. Carbohydrate Polymers 127: 264–274 (2015) 5 Wei CX, Qin FL, Zhou WD, Xu B, Chen C, Chen, YF, Wang YP, Gu MH, Liu QQ, Comparison of the crystalline properties and structural changes of starches from high-amylose transgenic rice and its wild type during heating. Food Chemistry 128: 645–652 (2011) 6 Teng B, Zhang Y, Du SY, Wu JD, Li ZF, Luo ZX, Yang JB, Crystalline, thermal and swelling properties of starches from single-segment substitution lines with different Wx alleles in rice (Oryza sativa L.). J Sci Food Agric 97: 108–114 (2017) 7 Hizukuir S, Polymodal dsitrbution of the chain lengths of amylopectins, and its significance. Carbohydr Res 147: 342–347 (1986) 8 Yuan RC, Thimpson DB, Bover CD, Fine structure of amylopectin in relation to gelatinization and retrogradation behavior of maize starches from three wx-containing genotypes in two inbred lines. Cereal Chem 70 (1): 81–89 (1993) 9 Kong XL, Chen YL, Zhu P, Sui ZQ, Corke H, Bao JS, Relationships among Genetic, Structural, and Functional Properties of Rice Starch. J. Agric. Food Chem 63: 6241−6248 (2015) 10 Nakamura Y, Sato A, Juliano BO, Short-Chain-Length Distribution in Debranched
This article is protected by copyright. All rights reserved.
Rice Starches Differing in Gelatinization Temperature or Cooked Rice Hardness. Starch/Stärke 58: 155–160 (2006)
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11 Nakamura Y, Sakurai A, Inaba Y, Kimura K, Iwasawa N, Nagamine T, The fine structure of amylopectin in endosperm from Asian cultivated rice can be largely classified into two classes. Starch/Starke 54: 117–131 (2002) 12 Macdonald FD, Preiss J, Partial Purification and Characterization of Granule-Bound Starch Synthases from Normal and Waxy Maize. Plant Physiol 78: 849–852 (1985) 13 Hirano HY, Eiguchi M, Sano Y, A single base change alter the regulation of the waxy gene at the posttranscriptional level during the domestication of rice. Mol. Bio. Evol 15: 978–98 (1998) 14 Hirano HY, Eiguchi M, Sano Y, Comparision of waxy gene regulation in the endosperm and pollen in Oryza satival L. Genes Genet Syst 75: 245–249 (2000) 15 Zhou LJ, Sheng WT, Wu J, Zhang CQ, Liu QQ, Deng QY, Differential expressions among five Waxy alleles and their effect on the eating and cooking qualities in specialty rice cultivars. Journal of Integrative Agriculture 14(6): 1153–1162 (2015) 16 Umemoto T, Horibata T, Aoki N, Hiratsuka M, Yano M, Inouchi N, Effects of Variation in Starch Synthase on Starch Properties and Eating Quality of Rice. Plant Prod. Sci 11: 472–480 (2008) 17 Jungro L, Zhang JM, Wang H, Li MB, Piao ZZ, Differences in Amylopectin Structure and Grain Quality of Rice Between Some High-Quality Japonica Cultivars from the Lower Yangtze River Region, China and Koshihikari from
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Niigata, Japan. Chin J Rice Sci 24: 379–384 (2010) 18 Cai YX, Wang W, Zhu ZW, Zhang ZJ, Yang JC, Zhu QS, The Physiochemical
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Characteristics of Amylopectin and Their Relationships to Pasting Properties of Rice Flour in Different Varieties. Scientia Agricultura Sinica 39: 1122–1129 (2006) 19 Cheetham NWH, Tao L, Variation in crystalline type with amylose content in maize starch granules: an X-ray powder diffraction study. Carbohydrate Polymers 36: 277–284 (1998) 20 Zhu DW, Zhang HC, Guo BW, Xu K, Dai QG, Wei CX, Zhou GS, Huo ZY, Effects of nitrogen level on structure and physicochemical properties of rice starch. Food Hydrocolloids 63: 525–532 (2017) 21 Zhu DW, Zhang HC, Guo BW, Xu K, Dai QG, Wei CX, Wei HY, Gao H, Hu YJ, Cui PY, Huo ZY, Effect of Nitrogen Management on the Structure and Physicochemical Properties of Rice Starch. J. Agric. Food Chem 64: 8019–8025 (2016) 22 Yang XY, Bi JG, Gilbert RG, Li GH, Liu ZH, Wang SH, Ding YF, Amylopectin chain length distribution in grains of japonica rice as affected by nitrogen fertilizer and genotype. Journal of Cereal Science 71: 230–238 (2016) 23 Gu JF, Chen J, Chen L, Wang ZQ, Zhang H, Yang JC, Grain quality changes and responses to nitrogen fertilizer of japonica rice cultivars released in the Yangtze River Basin from the 1950s to 2000s. The Crop Journal 14: 285–297 (2015) 24 Singh N, Pal N, Mahajan G, Singh S, Shevkani K, Rice grain and starch properties: Effects of nitrogen fertilizer application. Carbohydrate Polymers 86: 219–225
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(2011) 25 Duan H, Fu L, Ju CX, Liu LJ, Yang JC, Effects of Application of Nitrogen as
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Panicle-promoting Fertilizer on Seed Setting and Grain Quality of Rice under High Temperature Stress. Chin J Rice Sci 27: 591–602 (2013) 26 Ministry of Agriculture of the People's Republic of China. NY147-88 Evaluation Method of Rice Quality. Beijing: China Standard Press, 1988: 4–6. 27 Gao WW, Chen SP, Wang LP, Chen LK, Guo T, Wang H, Chen ZQ. Association Analysis of Rice Cooking Quality Traits with Molecular Markers. Scientia Agricultura Sinica 50: 599–611 (2017) 28 Teng B, Zhang Y, Wu JD, Cong XH, Wang RY, Han YH, Luo ZX, Association between allelic variation at the Waxy locus and starch physicochemical properties using single-segment substitution lines in rice (Oryza sativa L.). Starch/Stärke 65: 1069–1077 (2013) 29 Kong XL, Zhu P, Sui ZQ, Bao JS, Physicochemical properties of starches from diverse rice cultivars varying in apparent amylose content and gelatinisation temperature combinations. Food Chemistry 172: 433–440 (2015) 30 Zhang ZJ, Li M, Fang YW, Liu FC, Lu Y, Meng QC, Peng JC, Yi XH, Gu MH, Yan CJ, Diversification of the Waxy Gene is Closely Related to Variations in Rice Eating and Cooking Quality. Plant Mol Biol Rep 30: 462–469 (2012) 31 Zhao J, Research on Wx Near-isogenic Line Rice Yields, Rice Quality and Effect of Panicle Fertilizer Application. Yangzhou University. (2013) 32 Jang EH, Lee SJ, Hong JY, Chung HJ, Lee YT, Kang BS, Lim ST, Correlation
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between physicochemical properties of japonica and indica rice starches. LWT-Food Science and Technology 66: 530–537 (2016)
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33 Cai RG, Zhang M, Han T, Xie XY, Xu CL, Gu F, Effect of Plant Density on Starch Content and Crystal Property of Wheat Grain. Journal of Triticeae Crops 34: 78–83 (2014) 34 Zhong LJ, Cheng FM, Zhang GP, Sun ZX, Differences in Starch Chain Length Distribution and Structure Characteristics of Early-indica Rice Under Different Temperature Treatments During Grain-filling. Scientia Agricultura Sinica 4: 272– 276 (2005) 35 Zhu DW, Zhang HC, Guo BW, Xu K, Dai QG, Wei CX, Zhou GS, Huo ZY, Effects of nitrogen level on structure and physicochemical properties of rice starch. Food Hydrocolloids 63: 525–532 (2017) 36 Cai JW, Man JM, Huang J, Liu QQ, Wei WX, Wei CX, Relationship between structure and functional properties of normal rice starches with different amylose contents. Carbohydrate Polymers 125: 35–44 (2015)
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Table Table 1 Basic information regarding the near-isogenic line Number
Materials
Genotype
Waxy
Amylose content (%)
Y1
Yangfunuo 4 ×Minghui 63
Wxb
Non-waxy
12.5
Y3 Y5 Y7
Yangfunuo 4 ×Teqing Yangfunuo 4 ×IR36 Yangfunuo 4 ×IR64
Wxa Wxa Wxin
Non-waxy Non-waxy Non-waxy
27.5 28.4 24.9
Y9
Yangfunuo 4
wx
waxy
2.4
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Table 2 Apparent amylose content and gel consistency of rice in different Wx genotypes Apparent amylose content
Gel consistency（mm）
Fertilizer treatment
Tested materials
2015
2016
2015
2016
N1
Y1 Y3 Y5 Y7 Y9
16.0 c 24.5 a 25.5 a 22.5 b 3.4 d
15.4 d 27.0 b 28.4 a 23.2 c 4.4 e
94.0 b 73.5 d 68.5 d 87.0 c 113.5 a
102.0 b 75.5 d 73.0 d 84.5 c 110.5 a
Y1
17.5 c
15.8 d
86.5 b
93.0 b
Y3
25.1 a
27.5 b
55.5 d
59.0 d
Y5
26.6 a
28.6 a
51.0 d
57.5 d
Y7
23.0 b
25.2 c
67.0 c
71.5 c
N2
N3
（%）
Y9
3.8 d
5.1 e
112.0 a
113.0 a
Y1
17.9 c
18.4 d
85.0 b
84.5 b
Y3
27.7 ab
30.3 b
38.5 d
42.5 d
Y5
28.5 a
31.5 a
38.0 d
41.0 d
Y7
23.8 b
26.9 c
62.5 c
60.0 c
Y9
4.2 d
5.3 e
106.0 a
109.5 a
N1: 0 kg N ha–1, N2: 60 kg N ha–1, N3: 120 kg N ha–1. Y1, Wxb genotype; Y3 and Y5, Wxa genotype; Y7, Wxin genotype; and Y9, wx genotype. Values with different letters are significantly different at p=0.05 level under the same treatment but different materials.
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Table 3 Response of RVA profile values of different Wx genotypes to late-stage
Year
2015
2016
fertilization
Fertilizer
Tested
treatment
materials
N1
N2
N3
N1
N2
N3
PKV
（cp)
HPV
BDV
CPV
SBV
CSV
PaT
PeT
(cp)
(cp)
(cp)
(cp)
(cp)
(℃）
（min)
78.7 ab
5.6 b
Y1
3516 a
1653 b
1863 a
2912 d
-604 c
1259 c
Y3
3259 b
1998 a
1261 c
3572 a
313 a
1574 a
Y5
2685 c
1549 bc
1137 d
3031 c
346 a
1483 ab
Y7
3567 a
1887 a
1680 b
3295 b
-273 b
1408 b
Y9
3213 b
1520 c
1693 b
1963 e
-1250 d
443 d
Y1
3492 a
1689 bc
1803 a
2896 d
-596 c
1207 c
79.9 a
5.7 b
Y3
3014 d
2038 a
976 d
3559 a
545 a
1521 a
77.1 b
6.0 a
Y5
2754 e
1704 b
1050 d
3251 c
497 a
1547 a
77.9 b
5.8 b
Y7
3372 b
1937 a
1435 c
3360 b
-12 b
1423 b
77.1 b
5.8 b
Y9
3220 c
1579 c
1641 b
2019 e
-1201 d
440 d
79.2 a
4.2 c
Y1
3433 a
1693 b
1740 a
2876 b
-557 c
1183 b
79.9 a
5.8 b
Y3
2951 c
2029 a
923 d
3459 a
508 a
1431 a
77.9 c
6.1 a
Y5
2334 d
1383 d
951 d
2759 c
426 a
1376 a
77.5 c
5.8 b
Y7
3371 a
2113 a
1258 c
3520 a
149 b
1407 a
78.3 bc
6.0 a
Y9
3145 b
1568 c
1577 b
1577 d
-1050 d
528 c
79.1 ab
4.3 c
Y1
3275 a
1568 b
1707 a
2510 d
-766 c
942 c
82.0 a
5.8 b
Y3
2888 c
2206 a
683 d
3684 b
796 a
1479 a
78.7 b
6.1 a
Y5
3025 b
2292 a
733 d
3787 a
763 a
1495 a
79.1 b
6.1 a
Y7
2736 d
1700 b
1036 c
2922 c
187 b
1223 b
77.9 b
5.9 b
Y9
2744 d
1332 c
1413 b
1694 e
-1050 d
363 d
80.6 a
4.3 c
Y1
3159 a
1605 c
1554 a
2620 d
-539 c
1015 c
81.5 a
5.9 b
Y3
2973 b
2295 a
678 c
3809 a
836 a
1514 a
78.3 b
6.2 a
Y5
2881 c
2318 a
563 d
3667 b
787 a
1349 b
79.5 b
6.3 a
Y7
2560 e
1872 b
689 c
2940 c
380 b
1068 c
79.1 b
6.2 a
Y9
2658 d
1339 d
1319 b
1705 e
-953 d
366 d
81.1 a
4.4 c
Y1
3126 a
1785 c
1341 a
2732 d
-395 c
947 b
82.7 a
6.0 b
Y3
2784 c
2258 a
526 d
3623 b
840 a
1365 a
78.6 c
6.4 a
Y5
2950 b
2393 a
558 cd
3765 a
815 a
1372 a
79.1 c
6.4 a
Y7
2692 d
2042 b
650 c
3085 c
393 b
1043 b
78.6 c
6.3 a
Y9
2625 d
1411 d
1215 b
1787 e
-839 d
376 c
81.2 b
4.4 c
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76.7 d
5.8 a
77.1 cd
5.6 b
77.9 bc
5.6 b
79.1 a
4.2 c
N1: 0 kg N ha–1, N2: 60 kg N ha–1, N3: 120 kg N ha–1. Y1, Wxb genotype; Y3 and Y5, Wxa genotype; Y7, Wxin genotype; and Y9, wx genotype. PKV, peak viscosity; HPV,
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hot paste viscosity; BDV, breakdown viscosity; CPV, cool paste viscosity; SBV, setback viscosity; CSV, consistency viscosity; PaT, pasting temperature; PeT, Peak time. Values with different letters are significantly different at p=0.05 level under the same treatment but different materials.
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Table 4 Response of the starch structure of different rice Wx genotypes to late-stage fertilization Amylopectin（％）
Amylose content （％）
Fertilizer treatment
Tested materials
Short chain
Long chain
N1
Y1 Y3 Y5 Y7 Y9
58.61 b 54.06 c 52.57 d 57.47 b 76.02 a
19.56 b 19.23 b 18.96 b 19.38 b 23.93 a
21.82 d 26.71 b 28.47 a 23.15 c 0.05 e
3.00 ab 2.81 bc 2.77 c 2.97 bc 3.18 a
Y1
60.38 b
20.47 b
19.15 c
2.95 ab
Y3
52.63 d
19.06 c
28.31 a
2.76 bc
Y5
51.52 d
19.40 bc
29.07 a
2.66 c
Y7
56.10 c
19.88 bc
24.01 b
2.82 abc
Y9
75.13 a
24.87 a
0.01 d
3.02 a
Y1
61.19 b
21.23 b
17.58 d
2.88 ab
Y3
52.82 d
19.60 c
27.59 b
2.70 bc
Y5
49.96 e
19.55 c
30.48 a
2.56 c
N2
N3
Degree of branching
Y7
55.44 c
20.12 bc
24.43 c
2.76 bc
Y9
75.04 a
24.96 a
0.00 e
3.01 a
1946.599** 6.789** 7.851**
93.911** 6.184* 0.556
2976.551** 0.086 11.948**
19.505** 8.889** 0.278
Between the material Between fertilizer Interaction
N1: 0 kg N ha–1, N2: 60 kg N ha–1, N3: 120 kg N ha–1. Y1, Wxb genotype; Y3 and Y5, Wxa genotype; Y7, Wxin genotype; and Y9, wx genotype. Values with different letters are significantly different at p=0.05 level under the same treatment but different materials.*indicates significance at the p = 0.05 level; **indicates significance at the p = 0.01 level
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Table 5 Correlation analysis of RVA profile values, amylose content, and amylopectin branching degrees of different Wx genotypes PKV
Amylose content Degree of branching
HPV
BDV
CPV
-0.328
0.421
-0.624*
0.890**
0.929**
0.980**
0.668
-0.241
0.856
-0.626
-0.909
-0.753
**
**
SBV
*
CSV
**
PeT
PaT
**
-0.662**
0.942**
0.598
-0.743**
*
PKV, peak viscosity; HPV, hot paste viscosity; BDV, breakdown viscosity; CPV, cool paste viscosity; SBV, setback viscosity; CSV, consistency viscosity; PaT, pasting temperature; PeT, Peak time.* means significant correlation at the p=0.05 level; ** means significant correlation at the p=0.01 level.
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Table 6 Response of the relative crystallinity of different Wx genotypes of starch to late-stage fertilization Fertilizer treatment
N1
N2
N3
Tested materials
Relative crystallinity（%）
Y1
32.2 a
Y3
28.5 b
Y5
28.2 b
Y7
29.8 ab
Y9
33.0 a
Y1
32.3 b
Y3
28.1 bc
Y5
27.0 c
Y7
29.7 bc
Y9
35.7 a
Y1
31.2 b
Y3
27.6 c
Y5
27.7 c
Y7
27.8 c
Y9
35.8 a
N1: 0 kg N ha–1, N2: 60 kg N ha–1, N3: 120 kg N ha–1. Y1, Wxb genotype; Y3 and Y5, Wxa genotype; Y7, Wxin genotype; and Y9, wx genotype. Values with different letters are significantly different at p=0.05 level under the same treatment but different materials.
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Table 7 Starch swelling characteristics and solubility of rice with different Wx genotypes Fertilizer treatment
N1
N2
Tested materials
Swelling Power（g/g)
Solubility（％）
Y1
19.38 ab
7.00 c
Y3
18.19 b
Y5
16.53 b
Y7
18.42 b
9.14 bc
Y9
23.84 a
42.52 a
15.61 b
Y1
19.35 ab
7.99 b
Y3
16.19 bc
12.88 b
Y5
15.54 c
Y7 Y9
15.81 b 17.59 bc
22.12 a
Y1 N3
10.30 bc
11.00 b 54.24 a
19.10 ab
8.48 b
Y3
15.45 b
11.81 b
Y5
14.56 b
15.83 b
Y7 Y9
17.00 ab 20.29 a
11.50 b 57.87 a
N1: 0 kg N ha–1, N2: 60 kg N ha–1, N3: 120 kg N ha–1. Y1, Wxb genotype; Y3 and Y5, Wxa genotype; Y7, Wxin genotype; and Y9, wx genotype. Values with different letters are significantly different at p=0.05 level under the same treatment but different materials.
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Table 8 Correlation of relative crystallinity and swelling potential of starch with amylose content and branching degrees of amylopectin Relative crystallinity Amylose content Degree of branching
-0.920** 0.837**
Swelling Power
Solubility
-0.902** 0.955**
0.825** -0.640*
* means significant correlation at the p=0.05 level; ** means significant correlation at the p=0.01 level.
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Figures
Figure 1 Distribution of branches and combinations of starch in different Wx genotypes N1: 0 kg N ha–1, N2: 60 kg N ha–1, N3: 120 kg N ha–1. Y1, Wxb genotype; Y3 and Y5, Wxa genotype; Y7, Wxin genotype; and Y9, wx genotype.
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Fig.2 X-ray diffraction patterns of different Wx genotypes N1: 0 kg N ha–1, N2: 60 kg N ha–1, N3: 120 kg N ha–1. Y1, Wxb genotype; Y3 and Y5, Wxa genotype; Y7, Wxin genotype; and Y9, wx genotype.
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XianMei Cao, HuiYan Sun, ChunGe Wang, XiaoJia Ren, HongFei Liu, ZuJian Zhang*
Jiangsu Key Laboratory of Crop Genetics and Physiology/Key Laboratory of Plant Functional Genomics, Ministry of Education of China, Agricultural College of Yangzhou University, 225009, China
*
For
corresponding
(Zujian
Zhang;
Tel.:
+86-514-87972133;
Fax:
+86-514-97979317; Email: [email protected]
Abstract BACKGROUND: With the rapid development of modern agriculture, high-quality rice production and consumption has become the current urgent demand for the development of rice production. RESULT: In this paper, the effects of late-stage nitrogen fertilizer application on rice quality were studied under the same genetic background, Wx near-isogenic lines were used as test materials to study the starch composition, amylopectin structure, and cooking quality of rice. Results showed that rice amylose content and gel consistency This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.8723
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significantly differed when different Wx genes were tranformed into waxy rice, the law of apparent amylose content in rice is Wxa>Wxin>Wxb>wx at the same nitrogen
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level, while the trend of gel consistency was opposite to that of apparent amylose content, presenting obvious characteristics of Indica and Japonica varieties. As the amount of fertilizer application increased, apparent amylose content increased, gel consistency decreased, breakdown and peak viscosities dropped, and setback viscosity and peak time increased. Moreover, the cooking quality of rice significantly decreased with the use of nitrogen fertilizer, especially under low-level nitrogen fertilizer application. Amylopectin structure varied significantly in different genotypes of the Wx gene, and the degree of branching was as follows: wx>Wxb>Wxin>Wxa. This result indicated that the closer to Indica rice, the less short chains of amylopectin. Starch crystallinity and swelling potential were negatively correlated with amylose content but significantly positively correlated with amylopectin branching degree, decreasing with the increase of late-stage nitrogen fertilization. CONCLUSION: the late-stage nitrogen fertilization reduced the cooking quality of rice by increasing amylose content, reducing amylopectin branching degree, which decreased starch crystallinity, and aggravated pasting properties. Obviously, controlling late nitrogen application is essential to optimize rice quality. Key words: rice, Wx gene, nitrogen fertilizer, cooking quality, amylose, amylopectin 1. Introduction Starch weight accounts for 85%–90% of polished rice, which is mainly composed of amylose and amylopectin at 0%–30% and 70%–95% of rice starch, This article is protected by copyright. All rights reserved.
respectively.1-3 Amylose is a linear glucan chain with alpha-D-glucose units connected by the alpha-1,4-glycosidic bond, unbranched or rarely branched. The chain length is
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in the range of 1000–6000 DP. The amylopectin molecules in the branch are connected with alpha-1,4-glycosidic bond; the branches are connected with alpha-1,6-glycosidic bond, and the average branch length is 20–25 DP.4 Starch is lined with amylose and amylopectin molecules, with an alternating layer of crystalline and amorphous areas. The crystalline area constitutes a fixed layer of starch granules, and the amorphous area constitutes a sparse layer of starch granules. These areas are lined alternately.5,6 Hizukuri pointed out that amylopectin branches are not random but are clustered. Starch properties are determined not only by the amylose content but also by the distribution pattern of various branches of the cluster structure.7 The fine structure of amylopectin mainly includes the average polymerization degree, branching degree, average chain length, and chain length distribution.8,9 The average polymerization degree is 8200–12800. In general, the average polymerization degree in Indica rice is lower than that in Japonica rice. Higher branching degree of starch facilitates easy gelatinization of starch.10 Nakamura et al. showed that the structure of rice cultivars can be classified according to the different chain length distributions of amylopectin: L and S types. The L type possesses less short-length chain (DP≤11) and more medium-long length chain （DP=12–24）than the S type. Most Indica varieties belong to the L type.11 The amylose content in rice is mainly regulated by the Wx gene located on the short arm of the sixth chromosome.12 Different rice varieties including mainstream Indica, Japonica, tropical Japonica, and waxy varieties contain different
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Wx alleles. The Wx gene includes Wxa, Wxb, Wxin, and wx. Furthermore, nonwaxy gene Wx displays dominant expression on the waxy gene wx and evidently exerts a
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dose effect.13,14 Zhou et al. showed a similar rapid visco analyzer (RVA) profile for the five alleles of Wxa, Wxin, Wxb, Wxmq, and wx with a significant difference in amylose content. Nevertheless, the RVA value shows a significant difference.15 Umemoto et al. constructed the near-isogenic line using the genetic background of Nipponbare; the rice amylose content in the Wxa genotype is significantly higher than that in the parents, and the RVA spectrum characteristic values of peak viscosity (PKV) and breakdown viscosity (BDV) are significantly lower than those in the parents.16 In addition, amylopectin structure also exerts a remarkable effect on the cooking and eating qualities of rice. The amylose content of some high-quality Japonica Rice shows no significant difference with that of Koshihikari, but the ratio of short/long chain in amylopectin is higher in the former than in the latter.17 Cai et al. showed that the ratio of short chain part (Fr III) of branched chain starch is significantly positively correlated with PKV and BDV, but the long chain part (Fr (Ⅰ+Ⅱ)) displays an inverse correlation.18 Cheetham and Tao showed a strong correlation between the short/long chain ratio in amylopectin and starch crystallinity, i.e., higher ratio corresponds to higher crystallinity.19 However, the indirect effect of the Wx gene on starch structure is rarely reported. Nitrogen is an important and non-negligible environmental factor affecting rice quality. Although the effects of nitrogen fertilizer on the properties of rice starch were verified in numerous studies, combining the trends and observation is difficult.20-22
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Many studies suggested that nitrogen fertilizer is not contributing to rice quality improvement, but many research results showed that appropriate nitrogen can
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maintain and improve rice quality. Gu et al. suggested that the PKV, hot paste viscosity (HPV), cool paste viscosity (CPV), and BDV of starch decrease with increasing nitrogen fertilizer level; additionally, the eating quality is aggravated.23 Singh et al. reported that starch from rice grown with nitrogen application contains low amylose level.24 Duan concluded that middle or high nitrogen fertilizer level increases the short chain proportion of amylopectin and decreases the long chain proportion under water-saving conditions, thereby improving the quality of rice.25 The effect mechanism of nitrogen fertilizer application on rice synthesis is poorly understood. This mechanism is related to the test materials with complex genetic background. Conclusion vary due to the differences in varieties and environment used among studies. The present research used different Wx near-isogenic lines and prepared different fertilizer treatments to determine the effects of the Wx gene on rice quality and response characteristics of nitrogen under consistent genetic background. This study widens understanding of rice quality and provides the necessary theoretical basis for high-quality cultivation.
2. Materials and Methods 2.1 Test materials and growth conditions The experiment was carried out in 2015 and 2016 in the experimental field of the Agricultural College of Yangzhou University (32° 30＇N, 119° 30＇E). The condition
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of the test materials is shown in Table 1. Wx near-isogenic lines were used. Yangfunuo 4 was the recipient parents carrying four Indica rice cultivars (Minghui 63, Teqing,
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IR36, and IR64) with different Wx genes as donor parents; these lines were crossed and returned. The near-isogenic lines carrying different Wx genes were screened with the genetic background of Yannuo 4 through molecular marker-assisted selection. The soil in experimental site was a sandy loam with 23.2 g kg−1 organic matter, 98.2 mg kg−1 alkali-hydrolyzable N, 32.7 mg kg−1 Olsen-P, and 87.5 mg kg−1 exchangeable K. The previous crop was wheat.
The seeds were first sown in the paddy field on May
12. The seedlings were subsequently transplanted to the field on June 12 at a hill spacing of 20 × 18 cm with two seedlings per hill and harvested on October 6. Plot dimensions were 5 m × 3 m. Nitrogen treatment (N1: 0 kg N ha–1, N2: 60 kg N ha–1, N3: 120 kg N ha–1) was carried out seven days before heading, and each treatment was repeated three times. Basic fertilizer consisting of compound fertilizer (750 kg ha–1) and urea (150 kg ha–1) was applied uniformly. Tillering fertilizer was also evenly applied using urea (150 kg ha–1). The harvest was threshed and naturally dried. The seeds were stored for 3 months for rice quality determination. Rice powder was sieved over 100 mesh.
2.2 Gel consistency and amylose content analysis Gel consistency was determined in accordance with GB1350-1999. Amylose content was determined in accordance with the Ministry of Agriculture ministerial standard NY147-88.26
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2.3 Determination of starch gelatinization properties The viscosity tester RVA (Newport Scientific Instruments, Australia) was used in
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accordance with the American Society for Cereal Chemistry operating procedures (1995 61-02). The RVA spectrum characteristic value was mainly expressed by the peak viscosity (PKV), hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BDV, PKV–HPV), setback viscosity (SBV, CPV–PKV), consistence viscosity (CSV, CPV–HPV), peak time（PeT）, and pasting temperature (PaT).
2.4 Extraction of starch Approximately 10 g of rice flour was placed in a 50 mL centrifuge tube. Subsequently, NaOH solution (10-5 mol/L) was added to a 35 mL centrifuge tube; each tube contained 50 g kg-1 alkaline protease. After magnetic stirring under 42 °C constant temperature for 24 h, the homogenate was filtered through a 200 mesh sieve, and the filtrate was centrifuged at 4000 r min–1 for 20 min. The supernatant was discarded, and the yellow portion of the starch surface was removed. After centrifugation with deionized water, the extracts were centrifuged at 4000 r min-1 for 20 min. The above operation was repeated 3~5 times to remove ions and impurities. Afterward, 95% ethanol, chloroform/methanol mixture (V / V = 1: 1), and methanol/acetone mixture (V / V = 1: 1) were used to wash the extracts for 2~3 times and remove the fat. Baked until half dry, stirred with glass rods, starch was obtained by drying and screening over a 200-mesh sieve, then placed in a 4 °C refrigerator and
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sealed for storage.
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2.5 Determination of the branching degree of amylopectin For starch debranching treatment, 5 mg of starch was placed in a 2 mL centrifuge tube. Subsequently, 1.8 mL of DMSO/NaNO3 (0.5% W/W) was added. The mixture was placed at a constant temperature of 85 °C and dissolved at 33×g overnight. Centrifugation was performed at 1467×g for 10 min, and 1.5 mL of supernatant was carefully aspirated and placed in a 10 mL centrifuge tube. Afterward, anhydrous ethanol (four times of the volume) was added. The solution was mixed and centrifuged at 3724×g for 10 min. The supernatant was discarded, and the procedures were repeated three times. Approximately 1.0 mL of ultrapure water was added, and the solution was boiled for 1 h. Afterward, 0.9 mL of solution was obtained and placed in a 2 mL centrifuge tube; 0.1 mL of sodium acetate buffer (0.1 mol/L), 5 µL of sodium azide solution (0.04 g/mL), and 3.5 µL of amylase solution were also added. The resulting solution was vortexed and mixed at 37 °C overnight. About 180 µL of NaOH (0.1 mol/L) was added to adjust the pH of the solution to 7. For amylopectin GPC analysis, the off-branch of the sample was frozen with liquid nitrogen and placed in a freeze dryer. Approximately 1.8 mL of DMSO was added, and the temperature was set to 85 °C. The mixture was dissolved at 33×g overnight. After filtering at 0.45 µm, condensates were used for GPC (LP-GPC220, British PL). Three starch columns, namely, PL1110-6100, PL1110-6300, and PL1110-6525 were used for analysis.
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2.6 Starch grain type and crystallinity determination
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The starch was immersed in a sealed container containing a saturated aqueous solution of sodium chloride for one week and then subjected to an X-ray diffraction analyzer (D8 type multi-X diffractometer, Bruker AXS, Germany) under the following conditions: current, 40 mA; voltage, 40 kV; diffraction angle, 3°–40°; scanning speed, 0.3 s; and step size, 0.02°. The X-ray diffraction spectrum of the sample starch was measured twice a day. Origin75 software was used for mapping.
2.7 Determination of swelling potential and solubility A mixture of starch sample (30 mg) and ultrapure water (1 mL) in a 2 mL Ep tube (A) was place in shaking water bath at 90 °C for 1h. The mixture was subsequently centrifuged (4000×g) for 10min. The supernatant was placed in another 2 mL EP tube (B), and dried at 60 °C. The colloid adhering to the A pipe wall was weighed to obtain its water swelling weight. Solubility (%) = dissolved sample weight/ sample weight×100; Swelling power (g/g) = weight after water swelling/(sample weight – weight of dissolved sample).
2.8 Data processing Microsoft Excel software was used to organize data, and SPSS16.0 software was applied for statistical treatment.
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3. Results
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3.1 Effects of late-stage fertilization on the cooking quality of different Wx genotypes Table 2 shows that the apparent amylose content of rice is Y3, Y5 (Wxa)> Y7 (Wxin)> Y1 (Wxb)> Y9 (wx) at the same nitrogen level. This result showed significant rice starch formation among the Indica, Japonica, and waxy varieties during the two years. With the increase in late-stage fertilization level, the apparent amylose content increased, and the trend of gel consistency was opposite to that of apparent amylose content. The decreasing trend of gel consistency was more evident than that of amylose content. Reaction differences were observed among genotypes. The gel consistency of Y3, Y5 (Wxa genotype), and Y7 (Wxin genotype) rice varieties largely decreased 33.7 mm and 32.0 mm with the increase in fertilization levels in 2016. Such decline showed the sensitivity of rice gel consistency to late nitrogen fertilizer, and Y9 (wx genotype) rice remained essentially unchanged. Thus, in terms of cooking traits, Y3 and Y5 (Wxa genotype) obtained from Indica were relatively sensitive to late-stage fertilization, Y1 (Wxb genotype) was relatively insensitive, and Y9 (wx genotype) was insensitive. In addition, the amylose content showed significant difference in interannual, especially Y3, Y5 (Wxa genotype), and Y7 (Wxin genotype); this result indicated that these two genotypes were easily affected by climate factors.
Table 3 shows the response of the RVA profile of different Wx genotypes to
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late-stage fertilization. Among the different Wx genotypes, the BDV and PKV of Y1 (Wxb genotype) were the highest, and those of Y3 and Y5 (Wxa genotype) were
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relatively low. The CSV, SBV, and PeT of Y3 and Y5 (Wxa genotype) were relatively high, and those of Y9 (wx genotype) were the lowest; Y9 (wx gene type) and Y1 (Wxb genotype) showed high PaT of rice starch. With the increase in nitrogen fertilizer level in the late stage, the BDV and PKV were decreased, and the SBV and PeT of each material were increased, leading to cooking quality deterioration. The CSV and PaT were relatively stable. Considering the nitrogen fertilizer response differences among the genotypes, Y1 (Wxb genotype) and Y9 (wx genotype) were relatively insensitive, and Y3, Y5 (Wxa genotype), and Y7 (Wxin genotype) changed considerably, especially in 2015. Compared with varying from N1 to N2 levels, the amplitude of decrease in BDV and increase in SBV and PeT both were greater than those varied from N2 to N3 levels in all varieties. These results showed that starch cooking quality was sensitive to low nitrogen fertilizer.
3.2 Structural characteristics of amylopectin in rice with different Wx genotypes Figure 1 shows the molecular genotype of rice starch measured in Wx genotype distribution after isoamylase debranching. Except for waxy gene, the four genotypes exhibited typical three-peak distribution, but the peak level was significantly different. The peak area of Peak1 in the graph indicated the percentage of amylopectin short chain, the peak area of Peak2 indicated the percentage of long chain in amylopectin, and the peak area of Peak3 indicated amylose content. The ratio of Peak1/Peak2 is
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commonly regarded as the branching degree of amylopectin; higher ratio corresponds to higher amylopectin branching degree. Graphical data processing results are shown
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in Table 4.
Table 4 shows that Y9 (wx genotype) is a glutinous rice; it contains no amylose. The amylose content of Y3 and Y5 (Wxa genotype) rice was the highest. Distribution analysis on the amylopectin chain showed that short chain was proportional to the size of the trend of Y9 (wx)>Y1 (Wxb)>Y7 (Wxin)>Y3 and Y5 (Wxa). This result suggests that closeness to Indica corresponds to less short-chain proportion. With the increase in late-stage N level, except in Y1 (Wxb genotype), the percentage of short chain accounted in all genotypes exhibited a downward trend, whereas that of long chain was relatively the contrary; Y1 (Wxb genotype) also showed an evident increasing trend. The ratio of the length of the short chain to the middle-long chain represents amylopectin branching degree, and the branching degree decreased with increasing late-stage fertilization level. Variance analysis showed a significant difference in the short chain and branching degree of amylopectin under different fertilizer treatments, and their effects on amylose content exerted significant differences in materials. Therefore, late-stage fertilizer application significantly influenced the starch structure. A significant difference existed between the materials and fertilizers in the short chain of amylopectin, especially in the short-chain proportion of Y1 (Wxb genotype) responding to the fertilizer application, which were significantly different with other materials. The late-stage nitrogen fertilizer application resulted in decreased short
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chain proportion of Indica Wxa material and increased long chain proportion of
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Japonica Wxb material, which consequently reduced the starch branching degree.
Analysis on the amylose content, amylopectin branching degree and starch gelatinization characteristics (RVA profile) in Table 5 showed that the amylose content correlated negatively and negatively significantly with BDV and PaT (correlation coefficients: –0.624* and –0.662**), respectively. On the contrary, amylose content showed significantly positive correlation with the CPV, SBV, CSV, and PeT (correlation coefficients: 0.890**, 0.929**, 0.980**, and 0.942**, respectively). A significant positive correlation also existed between the branching degree of amylopectin and the PKV, BDV, and PaT; their correlation coefficients were 0.668**, 0.856**, and 0.598*, respectively. By contrast, significantly negative correlation existed between the amylopectin branching degree and the CPV, SBV, CSV, and PeT (correlation coefficients: –0.626*, –0.909**, –0.753**, and –0.743**, respectively). The amylose content or starch branching degree exhibited no significant correlation with HPV.
3.3 Crystallinity and swelling characteristics of rice starch with different Wx genotypes Figure 2 shows the X-ray diffraction patterns of the different genotypes of rice starch. The diffraction pattern of rice starch was all type A, and the 2θ angle was mainly at 15°, 17°, 18°, and 23°. This result indicated that the fertilization level
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exerted no influence on the crystal-type rice starch, but some differences existed in the peak intensity. The relative crystallinity in Table 6 showed that the relative
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crystallinity trend was Y9 (wx)> Y1 (Wxb)> Y7 (Wxin)> Y3 and Y5 (Wxa) at the same fertilizer level. Except for Y9 (wx), the relative crystallinity decreased with increasing nitrogen fertilizer level. Additionally, the decreasing range of Y7 (Wxin genotype) and Y3 and Y5 (Wxa genotype) rice was larger. The crystal formation of Wxin and Wxa rice starch was relatively sensitive to late-stage fertilizers.
The swelling potential and solubility of the rice starch for each genotype material are shown in Table 7. The water-soluble starch displayed advantages, such as water solubility, gelation, water retention, and swelling; these advantages were related to the cooking and eating quality of rice. The swelling power trend was Y9 (wx)> Y1 (Wxb)> Y7 (Wxin)> Y5 and Y3 (Wxa) at the same fertilizer level. Solubility showed a large value at Y9 (wx genotype), and the trend of others was opposite to that of starch swelling power. Swelling power decreased with increasing nitrogen fertilizer level. Y9 (wx genotype) rice decreased by 5.55%, and Y1 (Wxb genotype) was stable. Solubility increased with the application of nitrogen fertilizer. The increase of Y9 (wx genotype) was the highest at approximately 15.35%. The starch properties of Japonica materials were relatively stable in response to fertilizers.
Analysis results on the relationship of starch crystallinity and swelling properties or starch structure are shown in Table 8. The relative crystallinity and swelling power
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of starch showed a significant negative correlation with amylose content but a significant positive correlation with amylopectin branching degree. In terms of starch
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solubility, a significant positive correlation was found between solubility and amylose content, except for Y9 (wx genotype) rice, which showed a directly opposite trend to amylopectin branching degree.
3. Discussion 4.1 Performance characteristics of rice cooking quality in different Wx genotypes Amylose, PaT, and gel consistency are the main rice cooking quality evaluation index, and amylose content is of the most critical determinant.27-29 The Wx gene encodes the granule bound starch synthase in grains, which is responsible for the synthesis of amylose and the key gene to determine the amylose content of rice. The different Wx genotypes determine different rice cultivars. The different expression levels of the Wx gene in varieties cause the different amylose contents in the endosperm. The Wxa, Wxb, Wxin, and wx genotypes tested in this experiment are representatives of different rice genotypes. The different quality characteristics represent the performance of Indica, Japonica, tropical Japonica and glutinous rice types; these types were tested for several years, and they showed remarkably consistent performance.30,31 The change in amylose content caused adapted gel consistency and gelatinization temperature (Tables 2 and 3). In the RVA spectrum of visible characteristics, the RVA characteristics of the five materials also indicated the significant characteristics of Indica and Japonica. The BDV and PKV were the highest
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in the Wxb genotype, whereas relatively low in the Wxa genotype. The CSV, SBV, and PeT in the Wxa genotype were relatively high, whereas the lowest in wx. The test
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materials are of the same genetic background but with different near-isogenic lines of the Wx gene. Thus, the amylose content difference due to the Wx gene is the main reason for the different characteristics of the rice RVA. Jang et al. showed that Indica rice contains higher amylose, protein, and starch particles than Japonica rice, thereby showing higher gelatinization temperature at low viscosity. Additionally, amylose content and gelatinization temperature were positively correlated, but the PKV and BDV were significantly negatively correlated.32 However, in this study, the gelatinization temperature and amylose content were negatively related. Furthermore, rice quality is a quantitative trait that resulted from gene–environment interactions. Many previous studies using different varieties as test materials cannot exclude the influence of the genetic background, which may also be an important factor in varying research conclusions. Consequently, the quality of rice cultivation and regulation cannot be easily obtained. Given a new basis by using near-isogenic lines of molecular marker-assisted selection method to study the physiology and cultivation regulation, this method can not only analyze the biological effect of one specific gene, but also analyze the environmental effects without the interference of genetic background in variety, thereby obtaining a reliable conclusion. Correlation analysis determining the effect of the four Wx genotypes of five near-isogenic lines on the cooking quality of rice verified the significance of screening existing knowledge. 4.2 Chain length distribution of amylopectin in different Wx genotypes and their
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effects The Wx gene is the major gene that directly determines the amylose content.
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Different Wx alleles determine the difference between rice varieties. The results also showed that the Wx genotype of the different rice amylopectin chain length distribution exhibited differences. The short chain was proportional to the size with the following trend: wx>Wxb>Wxin>Wxa. The middle-long chains showed a trend opposite to that of the short chain. The trend of starch branching performance and short chain proportion was consistent; the trend changed from Indica to Japonica under the same genetic background, the amylose content declined, the amylopectin branching increased, and short chain increased. The Wx gene and amylopectin showed no evident change because no correlation existed between the amylopectin synthesis and biological function of the Wx gene. Therefore, the influence of the Wx gene on starch can be indirect. Starch synthesis in grains is an organic process. Each related gene and the corresponding enzyme form the whole network of starch synthesis and cause phase differences. For example, the Wx gene increases amylose content. Amylopectin adapts to the inevitable reduction in the formation of amylopectin, which also exerts a corresponding change degree and forms different amylopectin structures. In order to evaluate the indirect effect, only when Wx gene expression was the exclusive variable under premise of the same genetic background, can correct result be obtained. In addition, Wx gene synthesis effect on grain starch was mainly reflected in the amylose content and starch complex effect. The impact pathway should also be considered to further understand the mechanism of rice quality
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analysis. Starch is a natural crystalline polymer. Starch grain structure contains the crystal
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and amorphous phases of the two major components. Amylopectin mainly forms a crystalline zone, and amylose mainly forms an amorphous zone. The external environment cannot change the crystal type of starch, but it can change the crystallinity of starch.33-35 Results showed that the grain types of the Wx genotype were the same. Nevertheless, the relative crystallinity was wx> Wxb> Wxin> Wxa and that of Japonica rice was higher than that of Indica rice. Late-stage nitrogen application exerted no influence on the type of rice starch crystals, but it reduced the relative crystallinity. The relative crystallinity of rice starch was significantly negatively correlated with amylose content, while was significantly positively correlated with the branching degree of amylopectin. Hence, under uniform genetic background, crystallinity changes may be attributed to differences in starch composition and chain length structure and result in corresponding changes in starch swelling and solubility. Cai reported that the solubility of rice starch positively correlates with amylose content but negatively correlates with amylopectin short chain; rice starch swelling potential positively correlates with amylopectin and short chain but negatively correlates with amylose content.36 It is consistent with the results of this study. Furthermore, an inevitable correlation exists between rice starch crystallization characteristic and cooking quality. 4.3 Effects of nitrogen levels on the grain filling stage of starch structure and cooking and eating quality of rice
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The important role of nitrogen fertilizer in rice quality cannot be ignored. However, many conclusions regarding the effect of nitrogen fertilizer on rice quality
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are not consistent, which leads to the evaluation of the effect of nitrogen fertilizer on rice quality. In this study, we used the near-isogenic lines of the rice Wx gene with consistent genetic background. The different nitrogen fertilizer levels during grain filling were established through late-stage nitrogen fertilizer treatments. The responses of different Wx genes to nitrogen fertilizer level were also evaluated. Test results showed that the responses of wx, Wxb, Wxin, and Wxa to nitrogen fertilizer were mainly the same: with the increasing fertilization level, the apparent amylose content increased, the amylopectin branching ratio decreased, the crystallinity decreased, and the gel consistency decreased significantly. Consequently, the cooking quality of rice was sensitive to late-stage fertilizer application. RVA results also showed the same trend. In addition, low-level nitrogen fertilizer application can cause significant deterioration of the relevant gelatinization characteristics, which showed the sensitivity of rice quality to low-level nitrogen fertilizer. The negative effect of late nitrogen fertilizer application on rice quality was also significant. Inducing difference in starch synthesis by altering the Wx gene expression can affect rice cooking quality. The response intensity of the different Wx genotypes to nitrogen levels also exhibited differences. The Wxb and wx genotypes were relatively insensitive, and the Wxa and Wxin genotypes changed remarkably. The results showed that Indica rice and middle type rice were sensitive to nitrogen level, and Japonica rice was relatively stable. Therefore, the effect of N fertilization on rice quality differed among different rice
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varieties. Cultivation measures should also be adjusted accordingly. To date, nitrogen management has become a key cultivation measure in rice production to ensure high
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yield. However, ensuring high yield and quality is difficult during rice cultivation. China’s current production level is the world’s highest under the condition of the world’s highest level of fertilization. Balancing high quality and yield is a considerable challenge. The current results suggest that applying a high level of nitrogen fertilizer in the late stage decreases rice quality. Therefore, the late-stage application of nitrogen fertilizer should be controlled, and the high yield and high-quality cultivation technology system should be considered. The main technical approaches include the growth promotion during early stage, establishment of high-yield structure, and control of late-stage nitrogen fertilizer application to ensure high quality and strong growth. In addition, on the basis of controlling the application of nitrogen fertilizer in the late stage, appropriate water management measures, such as dry and wet irrigation are used to increase the root vigor and improve the growth activity of the rice filling and obtain high yield and quality. The effect of nitrogen application may also be reflected in the improved rice protein content and decreased eating quality. Hence, the effect of starch and protein syntheses on rice quality should be further investigated to determine the mechanism affecting rice quality.
5. conclusion Insertion of various Wx genes in waxy rice under the same genetic background resulted in significant differences in the amylose content and gel consistency of rice
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with different Wx genes, thereby showing evident characteristics of Indica and Japonica varieties. Moreover, amylopectin structure showed significant differences.
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The branching degree in different genotypes was wx>Wxb>Wxin>Wxa. This result showed that closeness to Indica resulted in less short chain proportion. Starch crystallinity and swelling power showed a corresponding change. Late-stage nitrogen fertilization increased amylose content and reduced significant amylopectin branching crystallinity, and gel consistency. Among the RVA characteristic spectrum, the BDV and PKV decreased, the SBV and PeT increased significantly, and the cooking quality significantly deteriorated. The Wxa and Wxin genotypes were more sensitive to late-stage fertilizer application than the Wxb and wx genotypes. These results suggest that the nitrogen fertilizer application exerts evident negative effect on the cooking quality of rice. Therefore, controlling the late-stage nitrogen application should be further studied.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (31371564)
References 1 Varatharajan V, Eric B, Structure-function relationships of starch components. Starch/Stärke 67: 55–68 (2015) 2 Kong XL, Chen YL, Zhu P, Sui ZQ, Corke H, Bao JS, Relationships among Genetic, Structural, and Functional Properties of Rice Starch. J. Agric. Food Chem 63:
This article is protected by copyright. All rights reserved.
6241−6248 (2015) 3 Patindol JA, Siebenmorgen TJ, Wang Y, Impact of environmental factors on rice
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starch structure: A review. Starch/Stärke 67: 42–54 (2015) 4 Wang K, Hasjim J, Wu AC, Li E, Henry RJ, Gilbert RG, Roles of GBSSI and SSIIa in determining amylose fine structure. Carbohydrate Polymers 127: 264–274 (2015) 5 Wei CX, Qin FL, Zhou WD, Xu B, Chen C, Chen, YF, Wang YP, Gu MH, Liu QQ, Comparison of the crystalline properties and structural changes of starches from high-amylose transgenic rice and its wild type during heating. Food Chemistry 128: 645–652 (2011) 6 Teng B, Zhang Y, Du SY, Wu JD, Li ZF, Luo ZX, Yang JB, Crystalline, thermal and swelling properties of starches from single-segment substitution lines with different Wx alleles in rice (Oryza sativa L.). J Sci Food Agric 97: 108–114 (2017) 7 Hizukuir S, Polymodal dsitrbution of the chain lengths of amylopectins, and its significance. Carbohydr Res 147: 342–347 (1986) 8 Yuan RC, Thimpson DB, Bover CD, Fine structure of amylopectin in relation to gelatinization and retrogradation behavior of maize starches from three wx-containing genotypes in two inbred lines. Cereal Chem 70 (1): 81–89 (1993) 9 Kong XL, Chen YL, Zhu P, Sui ZQ, Corke H, Bao JS, Relationships among Genetic, Structural, and Functional Properties of Rice Starch. J. Agric. Food Chem 63: 6241−6248 (2015) 10 Nakamura Y, Sato A, Juliano BO, Short-Chain-Length Distribution in Debranched
This article is protected by copyright. All rights reserved.
Rice Starches Differing in Gelatinization Temperature or Cooked Rice Hardness. Starch/Stärke 58: 155–160 (2006)
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11 Nakamura Y, Sakurai A, Inaba Y, Kimura K, Iwasawa N, Nagamine T, The fine structure of amylopectin in endosperm from Asian cultivated rice can be largely classified into two classes. Starch/Starke 54: 117–131 (2002) 12 Macdonald FD, Preiss J, Partial Purification and Characterization of Granule-Bound Starch Synthases from Normal and Waxy Maize. Plant Physiol 78: 849–852 (1985) 13 Hirano HY, Eiguchi M, Sano Y, A single base change alter the regulation of the waxy gene at the posttranscriptional level during the domestication of rice. Mol. Bio. Evol 15: 978–98 (1998) 14 Hirano HY, Eiguchi M, Sano Y, Comparision of waxy gene regulation in the endosperm and pollen in Oryza satival L. Genes Genet Syst 75: 245–249 (2000) 15 Zhou LJ, Sheng WT, Wu J, Zhang CQ, Liu QQ, Deng QY, Differential expressions among five Waxy alleles and their effect on the eating and cooking qualities in specialty rice cultivars. Journal of Integrative Agriculture 14(6): 1153–1162 (2015) 16 Umemoto T, Horibata T, Aoki N, Hiratsuka M, Yano M, Inouchi N, Effects of Variation in Starch Synthase on Starch Properties and Eating Quality of Rice. Plant Prod. Sci 11: 472–480 (2008) 17 Jungro L, Zhang JM, Wang H, Li MB, Piao ZZ, Differences in Amylopectin Structure and Grain Quality of Rice Between Some High-Quality Japonica Cultivars from the Lower Yangtze River Region, China and Koshihikari from
This article is protected by copyright. All rights reserved.
Niigata, Japan. Chin J Rice Sci 24: 379–384 (2010) 18 Cai YX, Wang W, Zhu ZW, Zhang ZJ, Yang JC, Zhu QS, The Physiochemical
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Characteristics of Amylopectin and Their Relationships to Pasting Properties of Rice Flour in Different Varieties. Scientia Agricultura Sinica 39: 1122–1129 (2006) 19 Cheetham NWH, Tao L, Variation in crystalline type with amylose content in maize starch granules: an X-ray powder diffraction study. Carbohydrate Polymers 36: 277–284 (1998) 20 Zhu DW, Zhang HC, Guo BW, Xu K, Dai QG, Wei CX, Zhou GS, Huo ZY, Effects of nitrogen level on structure and physicochemical properties of rice starch. Food Hydrocolloids 63: 525–532 (2017) 21 Zhu DW, Zhang HC, Guo BW, Xu K, Dai QG, Wei CX, Wei HY, Gao H, Hu YJ, Cui PY, Huo ZY, Effect of Nitrogen Management on the Structure and Physicochemical Properties of Rice Starch. J. Agric. Food Chem 64: 8019–8025 (2016) 22 Yang XY, Bi JG, Gilbert RG, Li GH, Liu ZH, Wang SH, Ding YF, Amylopectin chain length distribution in grains of japonica rice as affected by nitrogen fertilizer and genotype. Journal of Cereal Science 71: 230–238 (2016) 23 Gu JF, Chen J, Chen L, Wang ZQ, Zhang H, Yang JC, Grain quality changes and responses to nitrogen fertilizer of japonica rice cultivars released in the Yangtze River Basin from the 1950s to 2000s. The Crop Journal 14: 285–297 (2015) 24 Singh N, Pal N, Mahajan G, Singh S, Shevkani K, Rice grain and starch properties: Effects of nitrogen fertilizer application. Carbohydrate Polymers 86: 219–225
This article is protected by copyright. All rights reserved.
(2011) 25 Duan H, Fu L, Ju CX, Liu LJ, Yang JC, Effects of Application of Nitrogen as
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Panicle-promoting Fertilizer on Seed Setting and Grain Quality of Rice under High Temperature Stress. Chin J Rice Sci 27: 591–602 (2013) 26 Ministry of Agriculture of the People's Republic of China. NY147-88 Evaluation Method of Rice Quality. Beijing: China Standard Press, 1988: 4–6. 27 Gao WW, Chen SP, Wang LP, Chen LK, Guo T, Wang H, Chen ZQ. Association Analysis of Rice Cooking Quality Traits with Molecular Markers. Scientia Agricultura Sinica 50: 599–611 (2017) 28 Teng B, Zhang Y, Wu JD, Cong XH, Wang RY, Han YH, Luo ZX, Association between allelic variation at the Waxy locus and starch physicochemical properties using single-segment substitution lines in rice (Oryza sativa L.). Starch/Stärke 65: 1069–1077 (2013) 29 Kong XL, Zhu P, Sui ZQ, Bao JS, Physicochemical properties of starches from diverse rice cultivars varying in apparent amylose content and gelatinisation temperature combinations. Food Chemistry 172: 433–440 (2015) 30 Zhang ZJ, Li M, Fang YW, Liu FC, Lu Y, Meng QC, Peng JC, Yi XH, Gu MH, Yan CJ, Diversification of the Waxy Gene is Closely Related to Variations in Rice Eating and Cooking Quality. Plant Mol Biol Rep 30: 462–469 (2012) 31 Zhao J, Research on Wx Near-isogenic Line Rice Yields, Rice Quality and Effect of Panicle Fertilizer Application. Yangzhou University. (2013) 32 Jang EH, Lee SJ, Hong JY, Chung HJ, Lee YT, Kang BS, Lim ST, Correlation
This article is protected by copyright. All rights reserved.
between physicochemical properties of japonica and indica rice starches. LWT-Food Science and Technology 66: 530–537 (2016)
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33 Cai RG, Zhang M, Han T, Xie XY, Xu CL, Gu F, Effect of Plant Density on Starch Content and Crystal Property of Wheat Grain. Journal of Triticeae Crops 34: 78–83 (2014) 34 Zhong LJ, Cheng FM, Zhang GP, Sun ZX, Differences in Starch Chain Length Distribution and Structure Characteristics of Early-indica Rice Under Different Temperature Treatments During Grain-filling. Scientia Agricultura Sinica 4: 272– 276 (2005) 35 Zhu DW, Zhang HC, Guo BW, Xu K, Dai QG, Wei CX, Zhou GS, Huo ZY, Effects of nitrogen level on structure and physicochemical properties of rice starch. Food Hydrocolloids 63: 525–532 (2017) 36 Cai JW, Man JM, Huang J, Liu QQ, Wei WX, Wei CX, Relationship between structure and functional properties of normal rice starches with different amylose contents. Carbohydrate Polymers 125: 35–44 (2015)
This article is protected by copyright. All rights reserved.
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Table Table 1 Basic information regarding the near-isogenic line Number
Materials
Genotype
Waxy
Amylose content (%)
Y1
Yangfunuo 4 ×Minghui 63
Wxb
Non-waxy
12.5
Y3 Y5 Y7
Yangfunuo 4 ×Teqing Yangfunuo 4 ×IR36 Yangfunuo 4 ×IR64
Wxa Wxa Wxin
Non-waxy Non-waxy Non-waxy
27.5 28.4 24.9
Y9
Yangfunuo 4
wx
waxy
2.4
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Table 2 Apparent amylose content and gel consistency of rice in different Wx genotypes Apparent amylose content
Gel consistency（mm）
Fertilizer treatment
Tested materials
2015
2016
2015
2016
N1
Y1 Y3 Y5 Y7 Y9
16.0 c 24.5 a 25.5 a 22.5 b 3.4 d
15.4 d 27.0 b 28.4 a 23.2 c 4.4 e
94.0 b 73.5 d 68.5 d 87.0 c 113.5 a
102.0 b 75.5 d 73.0 d 84.5 c 110.5 a
Y1
17.5 c
15.8 d
86.5 b
93.0 b
Y3
25.1 a
27.5 b
55.5 d
59.0 d
Y5
26.6 a
28.6 a
51.0 d
57.5 d
Y7
23.0 b
25.2 c
67.0 c
71.5 c
N2
N3
（%）
Y9
3.8 d
5.1 e
112.0 a
113.0 a
Y1
17.9 c
18.4 d
85.0 b
84.5 b
Y3
27.7 ab
30.3 b
38.5 d
42.5 d
Y5
28.5 a
31.5 a
38.0 d
41.0 d
Y7
23.8 b
26.9 c
62.5 c
60.0 c
Y9
4.2 d
5.3 e
106.0 a
109.5 a
N1: 0 kg N ha–1, N2: 60 kg N ha–1, N3: 120 kg N ha–1. Y1, Wxb genotype; Y3 and Y5, Wxa genotype; Y7, Wxin genotype; and Y9, wx genotype. Values with different letters are significantly different at p=0.05 level under the same treatment but different materials.
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Table 3 Response of RVA profile values of different Wx genotypes to late-stage
Year
2015
2016
fertilization
Fertilizer
Tested
treatment
materials
N1
N2
N3
N1
N2
N3
PKV
（cp)
HPV
BDV
CPV
SBV
CSV
PaT
PeT
(cp)
(cp)
(cp)
(cp)
(cp)
(℃）
（min)
78.7 ab
5.6 b
Y1
3516 a
1653 b
1863 a
2912 d
-604 c
1259 c
Y3
3259 b
1998 a
1261 c
3572 a
313 a
1574 a
Y5
2685 c
1549 bc
1137 d
3031 c
346 a
1483 ab
Y7
3567 a
1887 a
1680 b
3295 b
-273 b
1408 b
Y9
3213 b
1520 c
1693 b
1963 e
-1250 d
443 d
Y1
3492 a
1689 bc
1803 a
2896 d
-596 c
1207 c
79.9 a
5.7 b
Y3
3014 d
2038 a
976 d
3559 a
545 a
1521 a
77.1 b
6.0 a
Y5
2754 e
1704 b
1050 d
3251 c
497 a
1547 a
77.9 b
5.8 b
Y7
3372 b
1937 a
1435 c
3360 b
-12 b
1423 b
77.1 b
5.8 b
Y9
3220 c
1579 c
1641 b
2019 e
-1201 d
440 d
79.2 a
4.2 c
Y1
3433 a
1693 b
1740 a
2876 b
-557 c
1183 b
79.9 a
5.8 b
Y3
2951 c
2029 a
923 d
3459 a
508 a
1431 a
77.9 c
6.1 a
Y5
2334 d
1383 d
951 d
2759 c
426 a
1376 a
77.5 c
5.8 b
Y7
3371 a
2113 a
1258 c
3520 a
149 b
1407 a
78.3 bc
6.0 a
Y9
3145 b
1568 c
1577 b
1577 d
-1050 d
528 c
79.1 ab
4.3 c
Y1
3275 a
1568 b
1707 a
2510 d
-766 c
942 c
82.0 a
5.8 b
Y3
2888 c
2206 a
683 d
3684 b
796 a
1479 a
78.7 b
6.1 a
Y5
3025 b
2292 a
733 d
3787 a
763 a
1495 a
79.1 b
6.1 a
Y7
2736 d
1700 b
1036 c
2922 c
187 b
1223 b
77.9 b
5.9 b
Y9
2744 d
1332 c
1413 b
1694 e
-1050 d
363 d
80.6 a
4.3 c
Y1
3159 a
1605 c
1554 a
2620 d
-539 c
1015 c
81.5 a
5.9 b
Y3
2973 b
2295 a
678 c
3809 a
836 a
1514 a
78.3 b
6.2 a
Y5
2881 c
2318 a
563 d
3667 b
787 a
1349 b
79.5 b
6.3 a
Y7
2560 e
1872 b
689 c
2940 c
380 b
1068 c
79.1 b
6.2 a
Y9
2658 d
1339 d
1319 b
1705 e
-953 d
366 d
81.1 a
4.4 c
Y1
3126 a
1785 c
1341 a
2732 d
-395 c
947 b
82.7 a
6.0 b
Y3
2784 c
2258 a
526 d
3623 b
840 a
1365 a
78.6 c
6.4 a
Y5
2950 b
2393 a
558 cd
3765 a
815 a
1372 a
79.1 c
6.4 a
Y7
2692 d
2042 b
650 c
3085 c
393 b
1043 b
78.6 c
6.3 a
Y9
2625 d
1411 d
1215 b
1787 e
-839 d
376 c
81.2 b
4.4 c
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76.7 d
5.8 a
77.1 cd
5.6 b
77.9 bc
5.6 b
79.1 a
4.2 c
N1: 0 kg N ha–1, N2: 60 kg N ha–1, N3: 120 kg N ha–1. Y1, Wxb genotype; Y3 and Y5, Wxa genotype; Y7, Wxin genotype; and Y9, wx genotype. PKV, peak viscosity; HPV,
Accepted Article
hot paste viscosity; BDV, breakdown viscosity; CPV, cool paste viscosity; SBV, setback viscosity; CSV, consistency viscosity; PaT, pasting temperature; PeT, Peak time. Values with different letters are significantly different at p=0.05 level under the same treatment but different materials.
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Table 4 Response of the starch structure of different rice Wx genotypes to late-stage fertilization Amylopectin（％）
Amylose content （％）
Fertilizer treatment
Tested materials
Short chain
Long chain
N1
Y1 Y3 Y5 Y7 Y9
58.61 b 54.06 c 52.57 d 57.47 b 76.02 a
19.56 b 19.23 b 18.96 b 19.38 b 23.93 a
21.82 d 26.71 b 28.47 a 23.15 c 0.05 e
3.00 ab 2.81 bc 2.77 c 2.97 bc 3.18 a
Y1
60.38 b
20.47 b
19.15 c
2.95 ab
Y3
52.63 d
19.06 c
28.31 a
2.76 bc
Y5
51.52 d
19.40 bc
29.07 a
2.66 c
Y7
56.10 c
19.88 bc
24.01 b
2.82 abc
Y9
75.13 a
24.87 a
0.01 d
3.02 a
Y1
61.19 b
21.23 b
17.58 d
2.88 ab
Y3
52.82 d
19.60 c
27.59 b
2.70 bc
Y5
49.96 e
19.55 c
30.48 a
2.56 c
N2
N3
Degree of branching
Y7
55.44 c
20.12 bc
24.43 c
2.76 bc
Y9
75.04 a
24.96 a
0.00 e
3.01 a
1946.599** 6.789** 7.851**
93.911** 6.184* 0.556
2976.551** 0.086 11.948**
19.505** 8.889** 0.278
Between the material Between fertilizer Interaction
N1: 0 kg N ha–1, N2: 60 kg N ha–1, N3: 120 kg N ha–1. Y1, Wxb genotype; Y3 and Y5, Wxa genotype; Y7, Wxin genotype; and Y9, wx genotype. Values with different letters are significantly different at p=0.05 level under the same treatment but different materials.*indicates significance at the p = 0.05 level; **indicates significance at the p = 0.01 level
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Table 5 Correlation analysis of RVA profile values, amylose content, and amylopectin branching degrees of different Wx genotypes PKV
Amylose content Degree of branching
HPV
BDV
CPV
-0.328
0.421
-0.624*
0.890**
0.929**
0.980**
0.668
-0.241
0.856
-0.626
-0.909
-0.753
**
**
SBV
*
CSV
**
PeT
PaT
**
-0.662**
0.942**
0.598
-0.743**
*
PKV, peak viscosity; HPV, hot paste viscosity; BDV, breakdown viscosity; CPV, cool paste viscosity; SBV, setback viscosity; CSV, consistency viscosity; PaT, pasting temperature; PeT, Peak time.* means significant correlation at the p=0.05 level; ** means significant correlation at the p=0.01 level.
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Table 6 Response of the relative crystallinity of different Wx genotypes of starch to late-stage fertilization Fertilizer treatment
N1
N2
N3
Tested materials
Relative crystallinity（%）
Y1
32.2 a
Y3
28.5 b
Y5
28.2 b
Y7
29.8 ab
Y9
33.0 a
Y1
32.3 b
Y3
28.1 bc
Y5
27.0 c
Y7
29.7 bc
Y9
35.7 a
Y1
31.2 b
Y3
27.6 c
Y5
27.7 c
Y7
27.8 c
Y9
35.8 a
N1: 0 kg N ha–1, N2: 60 kg N ha–1, N3: 120 kg N ha–1. Y1, Wxb genotype; Y3 and Y5, Wxa genotype; Y7, Wxin genotype; and Y9, wx genotype. Values with different letters are significantly different at p=0.05 level under the same treatment but different materials.
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Table 7 Starch swelling characteristics and solubility of rice with different Wx genotypes Fertilizer treatment
N1
N2
Tested materials
Swelling Power（g/g)
Solubility（％）
Y1
19.38 ab
7.00 c
Y3
18.19 b
Y5
16.53 b
Y7
18.42 b
9.14 bc
Y9
23.84 a
42.52 a
15.61 b
Y1
19.35 ab
7.99 b
Y3
16.19 bc
12.88 b
Y5
15.54 c
Y7 Y9
15.81 b 17.59 bc
22.12 a
Y1 N3
10.30 bc
11.00 b 54.24 a
19.10 ab
8.48 b
Y3
15.45 b
11.81 b
Y5
14.56 b
15.83 b
Y7 Y9
17.00 ab 20.29 a
11.50 b 57.87 a
N1: 0 kg N ha–1, N2: 60 kg N ha–1, N3: 120 kg N ha–1. Y1, Wxb genotype; Y3 and Y5, Wxa genotype; Y7, Wxin genotype; and Y9, wx genotype. Values with different letters are significantly different at p=0.05 level under the same treatment but different materials.
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Table 8 Correlation of relative crystallinity and swelling potential of starch with amylose content and branching degrees of amylopectin Relative crystallinity Amylose content Degree of branching
-0.920** 0.837**
Swelling Power
Solubility
-0.902** 0.955**
0.825** -0.640*
* means significant correlation at the p=0.05 level; ** means significant correlation at the p=0.01 level.
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Figures
Figure 1 Distribution of branches and combinations of starch in different Wx genotypes N1: 0 kg N ha–1, N2: 60 kg N ha–1, N3: 120 kg N ha–1. Y1, Wxb genotype; Y3 and Y5, Wxa genotype; Y7, Wxin genotype; and Y9, wx genotype.
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Fig.2 X-ray diffraction patterns of different Wx genotypes N1: 0 kg N ha–1, N2: 60 kg N ha–1, N3: 120 kg N ha–1. Y1, Wxb genotype; Y3 and Y5, Wxa genotype; Y7, Wxin genotype; and Y9, wx genotype.
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