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Relationships between transparency, amylose content, starch cavity, and moisture of brown rice kernels
Journal of Cereal Science 90 (2019) 102854
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Journal of Cereal Science journal homepage: http://www.elsevier.com/locate/jcs
Relationships between transparency, amylose content, starch cavity, and moisture of brown rice kernels Long Zhang a, b, Linglong Zhao a, b, Jing Zhang a, b, Xiuling Cai b, c, Qiaoquan Liu a, b, Cunxu Wei a, b, * a
Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Key Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou University, Yangzhou, 225009, China Co-Innovation Center for Modern Production Technology of Grain Crops of Jiangsu Province / Joint International Research Laboratory of Agriculture & Agri-Product Safety of the Ministry of Education, Yangzhou University, Yangzhou, 225009, China c National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Kernel transparency Moisture Amylose content Cavity of starch granule
Rice with low amylose content (AC) is popular due to the unique eating and cooking quality, but its transparency is poor. It is unclear why low AC rice has poor transparency until now. In this study, the transparency and microstructure of brown rice kernels were investigated in rices with different ACs. Chalkiness of endosperm resulted from the loosely packed starch granules, and existed in rice kernels with low and high moisture. However, dry kernels with low AC were opaque or dull due to the cavities in the center of starch granules, and could become transparent gradually with the increase of moisture. For rices with the same genetic background but different ACs, the cavity size was negatively correlated with AC, and the transparency of dry kernels was significantly correlated positively with AC and negatively with cavity size. The transparency of kernels was positively correlated with moisture of kernel, and the effect of moisture on transparency was significantly correlated positively with starch cavity size and negatively with kernel AC. The above results showed that the transparency of low AC rice was remarkably influenced by cavity size and AC of starch, and could be regulated through controlling the moisture of kernel.
1. Introduction Rice (Oryza sativa L.) is one of the world’s most important cereal crops and the staple food for more than half of the world’s population. With the improvement of living standards, the appearance, eating and cooking qualities of rice have been concerned more by consumers (Fitzgerald et al., 2009; Tian et al., 2009). An important way to improve the eating quality is to reduce the amylose content (AC) appropriately. Soft rice has low AC arranging from 2% to 15%, and exhibits the ad vantages of smooth, refreshing, elasticity and low degree of retrogra dation (Zhu et al., 2015). When soft rice is stored for a period of time after harvest, the kernels appear opaque or dull endosperm (Lu et al., 2018). The decreased transparency results in a poor appearance quality, and seriously affects the commercialization of soft rice. Therefore, it is very important and necessary to investigate the causes for the poor
transparency of soft rice. The transparency of rice kernels has been investigated in normal, hybrid, soft and glutinous rices. In normal Indica and Japonica rices, chalkiness is usually used to indicate the opaque region of the endo sperm and can be categorized as white-belly, white-back and white-core based on the region that become chalky (Guo et al., 2011; Xi et al., 2016). When amyloplast and/or protein body are poorly developed in rice endosperm cells during kernel development, incomplete grain filling occurs after grain maturation and dehydration. The irregular and loosely packed starch granules in endosperm cells result in the chalki ness of kernel (Wu et al., 2015). Wu et al. (2001) identified that the sterile lines and restorer lines of hybrid rice possess an opaque endosperm with low AC ranging from 1.8% to 14.5%. The opaque trait is related to the kernel moisture, starch structure, and starch morphology. The transparency of soft and
* Corresponding author. Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Key Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou University, Yangzhou, 225009, China. E-mail addresses: [email protected] (L. Zhang), [email protected] (L. Zhao), [email protected] (J. Zhang), [email protected] (X. Cai), cxwei@yzu. edu.cn (C. Wei). https://doi.org/10.1016/j.jcs.2019.102854 Received 2 September 2019; Received in revised form 6 October 2019; Accepted 6 October 2019 Available online 10 October 2019 0733-5210/© 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Appearance and microstructure of brown rice kernels with different ACs. (A, a, D, d, G, J), TQ; (B, b, E, e, H, K), NJ5055; (C, c, F, f, I, L), GLXN. (A–F), dry kernels; (a–f), rehydrated kernels. (D–F, d–f), cross fractured planes of kernels. The red arrows indicate the chalky region of rice kernel. (G–L), microstructure of starch granules in chalky (G–I) and nonchalky (J–L) regions under scanning electron microscope. The red arrows indicate the cavities in the center of starch granules. Scale bars ¼ 1 mm for (A–F, a–f) and 5 μm for (G–L). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
glutinous rice kernels is also closely related to the moisture of kernels. The soft rice varieties of Nanjing 46 and Guandong 194 have obviously dull endosperm under storage moisture below 12% (Li et al., 2018). Therefore, soft rice is usually sold in vacuum packaging to maintain a certain amount of moisture for improving its appear quality on the market (Li et al., 2018). Lu et al. (2018) have found that there are cavities in the center of starch granules in soft and glutinous rices. When rice endosperm con tains almost no amylose, it becomes glutinous rice and its endosperm is milky white and waxy (Zeng et al., 2007). The Japonica rice variety Nipponbare and its two near-isogenic lines Nip (Wxmp) and Nip (wx) contain 15.7%, 10.6% and 3.5% AC, respectively, and show no
observable differences in transparency and starch granule morphology before drying. While the endosperms of Nip (Wxmp) and Nip (wx) become opaque and waxy, respectively, and some cavities are observed in the center of the starch granules after drying (Li et al., 2018). Though some papers have showed that the transparency of soft rice is related to the moisture of kernel and the cavity of starch granule (Li et al., 2018; Lu et al., 2018), it is difficult to analyze the relationships between transparency, moisture, and starch cavity due to the different genetic backgrounds of rice varieties. In the previous study, we have developed some transgenic rice lines with different granule-bound starch synthase I (GBSSI) activities in a Japonica waxy rice back ground (Liu et al., 2015). Compared with wild type rice, these transgenic 2
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lines have significantly different ACs but the same amylopectin structure (Lin et al., 2018). In this study, we first investigated the transparency and micro structure of brown rice kernels of normal, soft and glutinous rices with different moistures, and then detailedly analyzed the relationships be tween transparency, AC, starch cavity, and moisture of brown rice kernels with different ACs but the same genetic background. This study could not only help us understand the poor transparency of low AC rice, but also provide important reference for appearance quality improve ment of low AC rice.
Nan-jing 5055 (NJ5055), Japonica glutinous rice variety Guang-lingxiang-nuo (GLXN), and three transgenic rice lines (E410D, Y268F and R408G) were used in this study. The E410D, Y268F, and R408G lines were derived from GLXN, and represent single amino acid substitutions at residues 410, 268, and 408 in the wild-type GBSSI protein, respec tively. The activity of GBSSI gradually decreases from R408G to Y268F and E410D (Liu et al., 2015). The GLXN, E410D line, Y268F line, and R408G line have 1.0%, 6.9%, 10.1%, and 16.2% AC, respectively (Lin et al., 2018). These rice materials were grown under normal agronomic practices in the same transgenic experimental field of Yangzhou University.
2. Materials and methods
2.2. Microscopy
2.1. Plant materials
The mature rice grains were dehusked with a rice huller (Huller JLGJ4.5, China). The brown rice kernels were photographed using a
The Indica normal rice variety Te-qing (TQ), Japonica soft rice variety
Fig. 2. Appearance and microstructure of brown rice dry kernels from GLXN and its transgenic rice lines with different ACs. (A), appearance of kernels under stereoscopic microscope. (B, C), microstructure of starch granules under scanning electron microscope. The kernels are transversely fractured by two tweezers (B) and polished by a glass knife (C). The regions indicated by red outline in the (B2) and (C2) are magnified in (B1) and (C1), respectively. The red arrows indicate the cavities in the center of starch granules. Scale bars ¼ 1 mm for (A, B1, C1) and 10 μm for (B2, C2). (D), the average area of cavity in the starch granule (n ¼ 20). The different letters indicate a significant difference (p < 0.05). (E), the relationship between cavity area and AC of starch. The “R” indicates the regression coefficient for linear fitting. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 3
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stereoscopic microscope (EZ4, Leica, Wetzla, Germany) under reflected and transmitted light. For the observation of naturally fractured plane, brown rice kernels were transversely fractured by two tweezers. For the observation of cavities in the interior of starch granules, the sample was clamped firmly in a block trimmer of Leica ultramicrotome and the fractured plane was smoothed with a glass knife following the method as described previously (Zhao et al., 2016). All the samples were mounted on aluminum specimen stubs with dual adhesive tape, coated with gold, and observed using a field emission scanning electron microscope (S-4800, Hitachi, Tokyo, Japan).
AC rice and waxy appearance of glutinous rice did not result from the chalkiness of kernels. In order to reveal the reasons for poor transparency of low AC rice kernels, the cross fractured plane of dry kernels were observed using scanning electron microscope (Fig. 1G–L). The chalky endosperms of TQ, NJ5055 and GLXN all had loosely packed starch granules with small size and some spherical shape (Fig. 1 G–I), a typical microstructure character of chalky endosperm of normal rice kernel (Chen et al., 2016; Li et al., 2014; Zhou et al., 2009). Though the nonchalky endosperms of TQ, NJ5055 and GLXN all had densely packed starch granules with polyhedral shape, significantly different microstructures were observed in the starch granules (Fig. 1J–L). The polygonal starch granules main tained intact morphology in nonchalky endosperm of TQ kernel, but the starch granules in nonchalky endosperm of NJ5055 and GLXN kernels are easily broken, and some cavities were observed in the center of granules. Similar phenomena have also been reported in soft rice (Lu et al., 2018). The present results indicated that the poor transparency of low AC rice kernels might result from the cavities of starch granules.
2.3. Rice transparency The brown rice kernels were photographed under transmitted light. Then, the color images were converted into black-and-white images with the desaturate tool in Adobe Photoshop CS6 Portable software and opened in ImageJ software. The polygon tool was used to draw the outline of brown rice. The gray scale of outline area in brown rice and image background was measured and recorded as G1 and G2, respec tively. The transparency (%) was obtained by G1/G2 � 100.
3.2. Appearance and microstructure of brown rice dry kernels with the same genetic background but different ACs
2.4. The dehydration treatment of brown rice
Although cavities have been observed in the broken starch granules of low AC rice kernels, it is difficult to quantify them due to the differ ences in genetic backgrounds between varieties. In the present study, we
For brown rice kernel dehydration, mature grains were freshly har vested without air drying and dehusked with a rice huller. The brown rice kernels (about 10 g) were packed in a net bag, and dried in an oven at 40 � C. The sample was weighted at 0, 4, 8, 12, 24, and 48 h after drying, and then grinded and passed through a 100-mesh sieve to measure the moisture. The experiments were repeated three times. 2.5. Determination of moisture The moisture of flour was determined using a halogen moisture analyzer (Mettler Toledo MJ33, Switzerland) following the method of Li et al. (2018). Briefly, 1 g of brown rice flour was evenly spread out in an aluminum foil dish, and dried at 120 � C for 15 min until the weight showed no change. 2.6. Statistical analysis Each experiment was repeated independently at least three times. All data reported in tables were means � standard deviations and analyzed by one-way analysis of variance (ANOVA) with Tukey’s test using SPSS version 16.0 Statistical Software. 3. Results 3.1. Appearance and microstructure of brown rice kernels with different ACs As the AC plays an important role in determining rice appearance quality, three rice varieties of TQ, NJ5055 and GLXN with different ACs and genetic backgrounds were selected in this study. The TQ, NJ5055, and GLXN have 22.7%, 10.4%, and 1.0% AC, respectively (Man et al., 2013; Wei et al., 2010; Zhu et al., 2016). The brown rice dry kernels were observed using a stereoscopic microscope (Fig. 1A–F). TQ kernels were transparent and had significantly white belly (Fig. 1A, D), NJ5055 kernels showed an opaque endosperm and had inconspicuous white belly (Fig. 1B, E), and GLXN kernels presented a milky waxy appearance and had imperceptible white belly (Fig. 1C, F). After brown rice kernels were immersed in H2O at 4 � C overnight, the appearance of TQ kernels did not change significantly (Fig. 1a, d), while NJ5055 kernels changed from opaque to transparent, and chalkiness was conspicuously detected (Fig. 1b, e); GLXN kernels changed from waxy to transparent, leading to that the white belly was clearly observed (Fig. 1c, f). The above results of dry and rehydrated kernels showed that the opaque appearance of low
Fig. 3. Transparency of brown rice dry kernels from GLXN and its transgenic rice lines with different ACs. (A), kernels under reflected light; (B), kernels under transmitted light; (C), moisture of kernels; (D), transparency of kernels; (E), the relationship between transparency and AC of kernels; (F), the rela tionship between transparency of kernels and cavity area of starch granules. Scale bar ¼ 1 mm (A, B). Data are means � standard deviations (n ¼ 3), and the different letters indicate a significant difference (p < 0.05) (C, D). The “R” in dicates the regression coefficient of linear fitting (E, F). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 4
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investigated the appearance and microstructure of dry kernels of GLXN and its derived three transgenic rice lines with the same genetic back ground and amylopectin structure but different ACs (Fig. 2). The cross fractured plane of kernels showed that brown rice kernels of GLXN and its three transgenic lines all had white-belly and nonchalky endosperm (Fig. 2A). For naturally fractured plane of kernels, starch granules in nonchalky endosperm of GLXN and E410D line had cavities in their interior, but no cavities were observed in the starch granules of non chalky endosperm in Y268F and R408G kernels due to that these starch granules were intact and not broken (Fig. 2B). In order to expose the inner structure of starch granules in Y268F and R408G kernels, the kernels were smoothly sectioned using a glass knife under ultramicro tome, and the starch granules were randomly cut to exhibit the micro structure of interior of starch granules. The cavities were clearly observed in the starch granules of nonchalky endosperm of GLXN, E410D, Y268F and R408G kernels (Fig. 2C). The size of cavity became smaller gradually from GLXN to E410D, Y268F and R408G (Fig. 2B, C, D). The linear fitting of AC and cavity area was analyzed among four rices (Fig. 2E). For four samples (n ¼ 4), the p is below 0.05 when the correlation coefficient (R) is over 0.950, and the p is below 0.01 when the R is over 0.990. In the present study, the R value ( 0.963) indicated that the area of cavity was significantly negatively correlated with the ACs (p < 0.05). The above results showed that starch granules with different ACs all had cavities in their interiors, but the starch granules with large cavities in glutinous and low AC rice were easily broken, and those with small cavities in high AC rice were difficultly broken.
3.3. Transparency of brown rice dry kernels with the same genetic background but different ACs The dry kernels of GLXN were waxy, E410D and Y268F presented opaque endosperms, and R408G had a transparent endosperm (Fig. 3A and B). Though the moisture of dry kernels showed no significant dif ferences among four rices (Fig. 3C), the transparency of kernels increased gradually from GLXN to E410D, Y268F and R408G (Fig. 3D). The transparency of dry kernel was significantly positively correlated with the AC (p < 0.05) (Fig. 3E) and negatively correlated with the cavity area of starch granules (p < 0.01) (Fig. 3F). Therefore, the transparency was influenced by the AC and cavity area of starch gran ules in rice endosperm. 3.4. Transparency and moisture of brown rice kernels with the same genetic background but different ACs during dehydration The freshly harvested brown rice kernels had high moisture. In order to clarify the effect of moisture on rice transparency, the freshly har vested brown rice kernels were oven-dried at 40 � C for different time. The freshly harvested kernels were transparent for GLXN, E410D, Y268F and R406G. During dehydration, the GLXN kernels gradually became waxy, the E410D, Y268F and R406G kernels gradually became opaque (Fig. 4). Though the transparency of kernels at the same dehydration time showed significant differences among four rice materials, they all
Fig. 4. Appearance of brown rice kernels from GLXN and its transgenic rice lines during dehydration at 40 � C. (A), kernels under reflected light; (B), kernels under transmitted light. Scale bar ¼ 1 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 5
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showed a decreasing trend with increase of dehydration time (Supple mental Table S1, Fig. 5A). The moisture of freshly harvested kernel was about 20%, and there were no significant differences among four rice materials. After dehydration for 48 h, the moisture of four lines was also similar (below 10%). However, the decreased rate of moisture was different among four rice lines during dehydration (Supplemental Table S2, Fig. 5B). The transparency was significantly positively corre lated with the moisture content for every rice material during dehy dration (p < 0.05 for GLXN, E410D and Y268F, and <0.01 for R408D) (Fig. 5C). Although there was a significant linear relationship between transparency and moisture of kernel, the slope constants (“K” values) of
linear fitting were significantly different among four rice materials (Fig. 5C). The “K” value represents the rate at which transparency changes with moisture content, and reflected the effect of moisture on transparency of kernel. The R408D line had significantly lowest “K” value (1.13), and the GLXN had the highest “K” value (2.14). The “K” value was significantly correlated negatively with AC (p < 0.05) and positively with the cavity area (p < 0.01) (Fig. 5D and E). These results showed that the transparency of kernel, especially for low AC rice, could be regulated through controlling the moisture of kernels, and the effect of moisture on transparency is influenced by AC and cavity size of starch granules.
Fig. 5. Transparency and moisture of brown rice kernels from GLXN and its transgenic rice lines during dehydration at 40 � C. (A), transparency; (B), moisture; (C), the relationship between transparency and moisture of brown rice kernels; (D), the relationship between “K” value and AC; (E), the relationship between “K” value and cavity area. The “K” value is the slope constant of linear fitting for transparency vs. moisture of kernels, and the “R” indicates the regression coefficient of linear fitting. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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4. Discussion
(Fig. 5D and E). In order to improve the appearance quality of kernel, soft rice on the market has high moisture in kernels (Lu et al., 2018). The present results showed that the transparency of kernels with low ACs could be regulated through controlling the moisture of kernels.
The rice transparency is an index for grading high-quality rice and has attracted a growing attention and interest. It is easier to observe and measure the transparency of kernels in polished rice than in brown rice. Therefore, the polished white rice, which is prepared through grinding the dry brown rice kernels with low moisture, is usually used to exhibit the transparency of rice kernels (Guo et al., 2011; Li et al., 2014, 2018; Lu et al., 2018; Wu et al., 2015). However, the freshly harvested brown rice kernels with high moisture are not suitable for preparing the pol ished rice. In fact, the transparency of brown rice can be detected and measured under both reflected and transmitted light, especially for the cross-section of kernel (Wu et al., 2001; Zeng et al., 2007). In order to investigate the relationship between transparency and moisture of rice kernels, we used the brown rice rather than the polished rice in the present study. Previous studies have described the decreased transparency as chalky, dull, milky white and opaque (Liu et al., 2009; Wan et al., 2005; Zhang et al., 2012). In this study, normal, soft and glutinous rice with different ACs were selected to further distinguish these phenotypes. All the three rice varieties contained chalky regions, but the chalky regions of soft and glutinous rice could be clearly seen only after rehydration of kernel (Fig. 1). The chalky regions of normal, soft and glutinous rices were packed loosely with starch granules having small size and spherical shape, and the nonchalky regions had polyhedral starch granules ar ranged closely in endosperm cells. Besides, many cavities were observed in the center of starch granules in the nonchalky regions of soft and glutinous rices (Fig. 1K and L). In conclusion, our results showed that chalkiness was not the cause for the decreased transparency of soft rice, and the cavities in the starch granules might be responsible for the poor transparency of dry kernels with low AC. Cavities have been observed in starch granules of soft rice Nanjing 46, Guandong 194 and Diantun 502 (Li et al., 2018; Lu et al., 2018). The starch granules of canna, potato and wheat also have cavities in their interior (Hall and Sayre, 1970, 1973; Baldwin et al., 1994). However, it is unclear what controls the size of cavity. In the present study, the glutinous rice GLXN and its derived three transgenic lines were selected to exclude the influence of different genetic backgrounds. Their starches have the same amylopectin structure, but significantly different ACs (Lin et al., 2018). The cavities could be observed in the broken starch gran ules of four rice materials, and their areas were significantly negatively correlated with the AC (Fig. 2). Normal starch granules are assembled by amylose and amylopectin. The amylose is concentrated mainly around the central hilum and the circumference of granule, and the amylopectin forms semicrystalline growth rings containing alternatively arranged crystalline and amorphous lamella (Cai et al., 2014; Glaring et al., 2006; Liu et al., 2019). The present results showed that AC was the critical factor determining the area of the cavity, and the lower the AC, the larger the cavity in the center of starch granules. Liu et al. (2009) have reported that three glutinous rice varieties with no AC show waxy phenotype and four Yunnan soft rice varieties with low AC (approximate 10%) show translucence. However, it is unclear for the relationship between AC and transparency of kernel. In the present study, linear regression analysis showed that the transparency was significantly positively correlated with the AC and negatively correlated with the area of cavity (Fig. 3E and F). Li et al. (2018) have found that the polished rice with 10.6% AC requires less time than the normal rice and more time than the glutinous rice to change from transparent to opaque during kernel drying. In the present study, the freshly harvested brown rice kernels changed from transparent to opaque in E410D, Y268F and R408G lines or waxy in GLXN during kernel dehydration (Fig. 4). Linear regression analysis showed that the transparency of kernel was positively correlated with moisture during dehydration, and the rate “K” value of transparency vs. moisture is different among four rice materials (Fig. 5C). Meanwhile, the “K” value was significantly correlated negatively with AC and positively with the cavity area
5. Conclusion Starch granules in brown rice dry kernels had cavities in their in teriors. The cavity size was negatively correlated with AC. A poor transparency of dry kernels with low AC was caused by the cavities of starch granules. The transparency of dry kernels with similar moisture was significantly correlated positively with AC and negatively with cavity size of starch granule. For kernels from the same rice variety, the transparency was significantly positively correlated with the moisture of kernels. The effect of moisture on transparency of kernel was correlated negatively with AC of kernel and positively with cavity size of starch granule. The transparency of low AC rice could be regulated through controlling the moisture of kernels. Declaration of competing interest The authors declare no conflict of interest. Acknowledgements This study was supported by grants from the Natural Science Foun dation of Jiangsu Province (BK20160461), the National Natural Science Foundation of China (31901427), the China Postdoctoral Science Foundation (2018T110561), the Talent Project of Yangzhou University, and the Priority Academic Program Development of Jiangsu Higher Education Institutions. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.jcs.2019.102854. References Baldwin, P.M., Adler, J., Davies, M.C., Melia, C.D., 1994. Holes in starch granules: confocal, SEM and light microscopy studies of starch granule structure. Starch 46, 341–346. Cai, C., Huang, J., Zhao, L., Liu, Q., Zhang, C., Wei, C., 2014. Heterogeneous structure and spatial distribution in endosperm of high-amylose rice starch granules with different morphologies. J. Agric. Food Chem. 62, 10143–10152. Chen, L., Gao, W., Chen, S., Wang, L., Zou, J., Liu, Y., Wang, H., Chen, Z., Guo, T., 2016. High-resolution QTL mapping for grain appearance traits and co-localization of chalkiness-associated differentially expressed candidate genes in rice. Rice 9, 48. Fitzgerald, M.A., McCouch, S.R., Hall, R.D., 2009. Not just a grain of rice: the quest for quality. Trends Plant Sci. 14, 133–139. Glaring, M.A., Koch, C.B., Blennow, A., 2006. Genotype-specific spatial distribution of starch molecules in the starch granule: a combined CLSM and SEM approach. Biomacromolecules 7, 2310–2320. Guo, T., Liu, X., Wan, X., Weng, J., Liu, S., Liu, X., Chen, M., Li, J., Su, N., Wu, F., Cheng, Z., Guo, X., Lei, C., Wang, J., Jiang, L., Wan, J., 2011. Identification of a stable quantitative trait locus for percentage grains with white chalkiness in rice (Oryza sativa). J. Integr. Plant Biol. 53, 598–607. Hall, D.M., Sayre, J.G., 1970. Internal architecture of potato and canna starch: Part I: crushing studies. Text. Res. J. 40, 147–157. Hall, D.M., Sayre, J.G., 1973. A comparision of starch granules as seen by both scanning electron and ordinary light microscopy. Starch 25, 119–123. Li, Q., Huang, L., Chu, R., Li, J., Jiang, M., Zhang, C., Fan, X., Yu, H., Gu, M., Liu, Q., 2018. Down-regulation of SSSII-2 gene expression results in novel low-amylose rice with soft, transparent grains. J. Agric. Food Chem. 66, 9750–9760. Li, Y., Fan, C., Xing, Y., Yun, P., Luo, L., Yan, B., Peng, B., Xie, W., Wang, G., Li, X., Xiao, J., Xu, C., He, Y., 2014. Chalk 5 encodes a vacuolar Hþ-translocating pyrophosphatase influencing grain chalkiness in rice. Nat. Genet. 46, 398–404. Lin, L., Zhang, L., Cai, X., Liu, Q., Zhang, C., Wei, C., 2018. The relationship between enzyme hydrolysis and the components of rice starches with the same genetic background and amylopectin structure but different amylose contents. Food Hydrocolloids 84, 406–413. Liu, D., Wang, W., Cai, X., 2015. Modulation of amylose content by structure-based modification of OsGBSS1 activity in rice (Oryza sativa L.). Plant Biotechnol. J. 12, 1297–1307.
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Liu, T., Ma, M., Guo, K., Hu, G., Zhang, L., Wei, C., 2019. Structural, thermal, and hydrolysis properties of large and small granules from C-type starches of four Chinese chestnut varieties. Int. J. Biol. Macromol. 137, 712–720. Liu, L., Ma, X., Liu, S., Zhu, C., Jiang, L., Wang, Y., Shen, Y., Ren, Y., Dong, H., Chen, L., Liu, X., Zhao, Z., Zhai, H., Wan, J., 2009. Identification and characterization of a novel Waxy allele from a Yunnan rice landrace. Plant Mol. Biol. 71, 609–626. Lu, Y., Zhang, X., Qi, Y., Zhang, C., Ling, Y., Liu, Q., 2018. Scanning electron microscopic analysis of grain cross-section from rice with different transparency. Chin. J. Rice Sci. 32, 189–199. Man, J., Yang, Y., Hang, J., Zhang, C., Chen, Y., Wang, Y., Gu, M., Liu, Q., Wei, C., 2013. Effect of simultaneous inhibition of starch branching enzymes I and IIb on the crystalline structure of rice starches with different amylose contents. J. Agric. Food Chem. 61, 9930–9937. Tian, Z., Qian, Q., Liu, Q., Yan, M., Liu, X., Yan, C., Liu, G., Gao, Z., Tang, S., Zeng, D., Wang, Y., Yu, J., Gu, M., Li, J., 2009. Allelic diversities in rice starch biosynthesis lead to a diverse array of rice eating and cooking qualities. Proc. Natl. Acad. Sci. U.S. A. 106, 21760–21765. Wan, X., Wan, J., Weng, J., Jiang, L., Bi, J., Wang, C., Zhai, H., 2005. Stability of QTLs for rice grain dimension and endosperm chalkiness characteristics across eight environments. Theor. Appl. Genet. 110, 1334–1346. Wei, C., Qin, F., Zhu, L., Zhou, W., Chen, Y., Wang, Y., Gu, M., Liu, Q., 2010. Microstructure and ultrastructure of high-amylose rice resistant starch granules modified by antisense RNA inhibition of starch branching enzyme. J. Agric. Food Chem. 58, 1224–1232. Wu, D., Xia, Y., Li, X., 2001. Formation basis of rice mist endosperm appearance and its rapid identifying factors. Chin. J. Rice Sci. 15, 192–196.
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Journal of Cereal Science journal homepage: http://www.elsevier.com/locate/jcs
Relationships between transparency, amylose content, starch cavity, and moisture of brown rice kernels Long Zhang a, b, Linglong Zhao a, b, Jing Zhang a, b, Xiuling Cai b, c, Qiaoquan Liu a, b, Cunxu Wei a, b, * a
Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Key Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou University, Yangzhou, 225009, China Co-Innovation Center for Modern Production Technology of Grain Crops of Jiangsu Province / Joint International Research Laboratory of Agriculture & Agri-Product Safety of the Ministry of Education, Yangzhou University, Yangzhou, 225009, China c National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Kernel transparency Moisture Amylose content Cavity of starch granule
Rice with low amylose content (AC) is popular due to the unique eating and cooking quality, but its transparency is poor. It is unclear why low AC rice has poor transparency until now. In this study, the transparency and microstructure of brown rice kernels were investigated in rices with different ACs. Chalkiness of endosperm resulted from the loosely packed starch granules, and existed in rice kernels with low and high moisture. However, dry kernels with low AC were opaque or dull due to the cavities in the center of starch granules, and could become transparent gradually with the increase of moisture. For rices with the same genetic background but different ACs, the cavity size was negatively correlated with AC, and the transparency of dry kernels was significantly correlated positively with AC and negatively with cavity size. The transparency of kernels was positively correlated with moisture of kernel, and the effect of moisture on transparency was significantly correlated positively with starch cavity size and negatively with kernel AC. The above results showed that the transparency of low AC rice was remarkably influenced by cavity size and AC of starch, and could be regulated through controlling the moisture of kernel.
1. Introduction Rice (Oryza sativa L.) is one of the world’s most important cereal crops and the staple food for more than half of the world’s population. With the improvement of living standards, the appearance, eating and cooking qualities of rice have been concerned more by consumers (Fitzgerald et al., 2009; Tian et al., 2009). An important way to improve the eating quality is to reduce the amylose content (AC) appropriately. Soft rice has low AC arranging from 2% to 15%, and exhibits the ad vantages of smooth, refreshing, elasticity and low degree of retrogra dation (Zhu et al., 2015). When soft rice is stored for a period of time after harvest, the kernels appear opaque or dull endosperm (Lu et al., 2018). The decreased transparency results in a poor appearance quality, and seriously affects the commercialization of soft rice. Therefore, it is very important and necessary to investigate the causes for the poor
transparency of soft rice. The transparency of rice kernels has been investigated in normal, hybrid, soft and glutinous rices. In normal Indica and Japonica rices, chalkiness is usually used to indicate the opaque region of the endo sperm and can be categorized as white-belly, white-back and white-core based on the region that become chalky (Guo et al., 2011; Xi et al., 2016). When amyloplast and/or protein body are poorly developed in rice endosperm cells during kernel development, incomplete grain filling occurs after grain maturation and dehydration. The irregular and loosely packed starch granules in endosperm cells result in the chalki ness of kernel (Wu et al., 2015). Wu et al. (2001) identified that the sterile lines and restorer lines of hybrid rice possess an opaque endosperm with low AC ranging from 1.8% to 14.5%. The opaque trait is related to the kernel moisture, starch structure, and starch morphology. The transparency of soft and
* Corresponding author. Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Key Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou University, Yangzhou, 225009, China. E-mail addresses: [email protected] (L. Zhang), [email protected] (L. Zhao), [email protected] (J. Zhang), [email protected] (X. Cai), cxwei@yzu. edu.cn (C. Wei). https://doi.org/10.1016/j.jcs.2019.102854 Received 2 September 2019; Received in revised form 6 October 2019; Accepted 6 October 2019 Available online 10 October 2019 0733-5210/© 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Appearance and microstructure of brown rice kernels with different ACs. (A, a, D, d, G, J), TQ; (B, b, E, e, H, K), NJ5055; (C, c, F, f, I, L), GLXN. (A–F), dry kernels; (a–f), rehydrated kernels. (D–F, d–f), cross fractured planes of kernels. The red arrows indicate the chalky region of rice kernel. (G–L), microstructure of starch granules in chalky (G–I) and nonchalky (J–L) regions under scanning electron microscope. The red arrows indicate the cavities in the center of starch granules. Scale bars ¼ 1 mm for (A–F, a–f) and 5 μm for (G–L). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
glutinous rice kernels is also closely related to the moisture of kernels. The soft rice varieties of Nanjing 46 and Guandong 194 have obviously dull endosperm under storage moisture below 12% (Li et al., 2018). Therefore, soft rice is usually sold in vacuum packaging to maintain a certain amount of moisture for improving its appear quality on the market (Li et al., 2018). Lu et al. (2018) have found that there are cavities in the center of starch granules in soft and glutinous rices. When rice endosperm con tains almost no amylose, it becomes glutinous rice and its endosperm is milky white and waxy (Zeng et al., 2007). The Japonica rice variety Nipponbare and its two near-isogenic lines Nip (Wxmp) and Nip (wx) contain 15.7%, 10.6% and 3.5% AC, respectively, and show no
observable differences in transparency and starch granule morphology before drying. While the endosperms of Nip (Wxmp) and Nip (wx) become opaque and waxy, respectively, and some cavities are observed in the center of the starch granules after drying (Li et al., 2018). Though some papers have showed that the transparency of soft rice is related to the moisture of kernel and the cavity of starch granule (Li et al., 2018; Lu et al., 2018), it is difficult to analyze the relationships between transparency, moisture, and starch cavity due to the different genetic backgrounds of rice varieties. In the previous study, we have developed some transgenic rice lines with different granule-bound starch synthase I (GBSSI) activities in a Japonica waxy rice back ground (Liu et al., 2015). Compared with wild type rice, these transgenic 2
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lines have significantly different ACs but the same amylopectin structure (Lin et al., 2018). In this study, we first investigated the transparency and micro structure of brown rice kernels of normal, soft and glutinous rices with different moistures, and then detailedly analyzed the relationships be tween transparency, AC, starch cavity, and moisture of brown rice kernels with different ACs but the same genetic background. This study could not only help us understand the poor transparency of low AC rice, but also provide important reference for appearance quality improve ment of low AC rice.
Nan-jing 5055 (NJ5055), Japonica glutinous rice variety Guang-lingxiang-nuo (GLXN), and three transgenic rice lines (E410D, Y268F and R408G) were used in this study. The E410D, Y268F, and R408G lines were derived from GLXN, and represent single amino acid substitutions at residues 410, 268, and 408 in the wild-type GBSSI protein, respec tively. The activity of GBSSI gradually decreases from R408G to Y268F and E410D (Liu et al., 2015). The GLXN, E410D line, Y268F line, and R408G line have 1.0%, 6.9%, 10.1%, and 16.2% AC, respectively (Lin et al., 2018). These rice materials were grown under normal agronomic practices in the same transgenic experimental field of Yangzhou University.
2. Materials and methods
2.2. Microscopy
2.1. Plant materials
The mature rice grains were dehusked with a rice huller (Huller JLGJ4.5, China). The brown rice kernels were photographed using a
The Indica normal rice variety Te-qing (TQ), Japonica soft rice variety
Fig. 2. Appearance and microstructure of brown rice dry kernels from GLXN and its transgenic rice lines with different ACs. (A), appearance of kernels under stereoscopic microscope. (B, C), microstructure of starch granules under scanning electron microscope. The kernels are transversely fractured by two tweezers (B) and polished by a glass knife (C). The regions indicated by red outline in the (B2) and (C2) are magnified in (B1) and (C1), respectively. The red arrows indicate the cavities in the center of starch granules. Scale bars ¼ 1 mm for (A, B1, C1) and 10 μm for (B2, C2). (D), the average area of cavity in the starch granule (n ¼ 20). The different letters indicate a significant difference (p < 0.05). (E), the relationship between cavity area and AC of starch. The “R” indicates the regression coefficient for linear fitting. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 3
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stereoscopic microscope (EZ4, Leica, Wetzla, Germany) under reflected and transmitted light. For the observation of naturally fractured plane, brown rice kernels were transversely fractured by two tweezers. For the observation of cavities in the interior of starch granules, the sample was clamped firmly in a block trimmer of Leica ultramicrotome and the fractured plane was smoothed with a glass knife following the method as described previously (Zhao et al., 2016). All the samples were mounted on aluminum specimen stubs with dual adhesive tape, coated with gold, and observed using a field emission scanning electron microscope (S-4800, Hitachi, Tokyo, Japan).
AC rice and waxy appearance of glutinous rice did not result from the chalkiness of kernels. In order to reveal the reasons for poor transparency of low AC rice kernels, the cross fractured plane of dry kernels were observed using scanning electron microscope (Fig. 1G–L). The chalky endosperms of TQ, NJ5055 and GLXN all had loosely packed starch granules with small size and some spherical shape (Fig. 1 G–I), a typical microstructure character of chalky endosperm of normal rice kernel (Chen et al., 2016; Li et al., 2014; Zhou et al., 2009). Though the nonchalky endosperms of TQ, NJ5055 and GLXN all had densely packed starch granules with polyhedral shape, significantly different microstructures were observed in the starch granules (Fig. 1J–L). The polygonal starch granules main tained intact morphology in nonchalky endosperm of TQ kernel, but the starch granules in nonchalky endosperm of NJ5055 and GLXN kernels are easily broken, and some cavities were observed in the center of granules. Similar phenomena have also been reported in soft rice (Lu et al., 2018). The present results indicated that the poor transparency of low AC rice kernels might result from the cavities of starch granules.
2.3. Rice transparency The brown rice kernels were photographed under transmitted light. Then, the color images were converted into black-and-white images with the desaturate tool in Adobe Photoshop CS6 Portable software and opened in ImageJ software. The polygon tool was used to draw the outline of brown rice. The gray scale of outline area in brown rice and image background was measured and recorded as G1 and G2, respec tively. The transparency (%) was obtained by G1/G2 � 100.
3.2. Appearance and microstructure of brown rice dry kernels with the same genetic background but different ACs
2.4. The dehydration treatment of brown rice
Although cavities have been observed in the broken starch granules of low AC rice kernels, it is difficult to quantify them due to the differ ences in genetic backgrounds between varieties. In the present study, we
For brown rice kernel dehydration, mature grains were freshly har vested without air drying and dehusked with a rice huller. The brown rice kernels (about 10 g) were packed in a net bag, and dried in an oven at 40 � C. The sample was weighted at 0, 4, 8, 12, 24, and 48 h after drying, and then grinded and passed through a 100-mesh sieve to measure the moisture. The experiments were repeated three times. 2.5. Determination of moisture The moisture of flour was determined using a halogen moisture analyzer (Mettler Toledo MJ33, Switzerland) following the method of Li et al. (2018). Briefly, 1 g of brown rice flour was evenly spread out in an aluminum foil dish, and dried at 120 � C for 15 min until the weight showed no change. 2.6. Statistical analysis Each experiment was repeated independently at least three times. All data reported in tables were means � standard deviations and analyzed by one-way analysis of variance (ANOVA) with Tukey’s test using SPSS version 16.0 Statistical Software. 3. Results 3.1. Appearance and microstructure of brown rice kernels with different ACs As the AC plays an important role in determining rice appearance quality, three rice varieties of TQ, NJ5055 and GLXN with different ACs and genetic backgrounds were selected in this study. The TQ, NJ5055, and GLXN have 22.7%, 10.4%, and 1.0% AC, respectively (Man et al., 2013; Wei et al., 2010; Zhu et al., 2016). The brown rice dry kernels were observed using a stereoscopic microscope (Fig. 1A–F). TQ kernels were transparent and had significantly white belly (Fig. 1A, D), NJ5055 kernels showed an opaque endosperm and had inconspicuous white belly (Fig. 1B, E), and GLXN kernels presented a milky waxy appearance and had imperceptible white belly (Fig. 1C, F). After brown rice kernels were immersed in H2O at 4 � C overnight, the appearance of TQ kernels did not change significantly (Fig. 1a, d), while NJ5055 kernels changed from opaque to transparent, and chalkiness was conspicuously detected (Fig. 1b, e); GLXN kernels changed from waxy to transparent, leading to that the white belly was clearly observed (Fig. 1c, f). The above results of dry and rehydrated kernels showed that the opaque appearance of low
Fig. 3. Transparency of brown rice dry kernels from GLXN and its transgenic rice lines with different ACs. (A), kernels under reflected light; (B), kernels under transmitted light; (C), moisture of kernels; (D), transparency of kernels; (E), the relationship between transparency and AC of kernels; (F), the rela tionship between transparency of kernels and cavity area of starch granules. Scale bar ¼ 1 mm (A, B). Data are means � standard deviations (n ¼ 3), and the different letters indicate a significant difference (p < 0.05) (C, D). The “R” in dicates the regression coefficient of linear fitting (E, F). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 4
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investigated the appearance and microstructure of dry kernels of GLXN and its derived three transgenic rice lines with the same genetic back ground and amylopectin structure but different ACs (Fig. 2). The cross fractured plane of kernels showed that brown rice kernels of GLXN and its three transgenic lines all had white-belly and nonchalky endosperm (Fig. 2A). For naturally fractured plane of kernels, starch granules in nonchalky endosperm of GLXN and E410D line had cavities in their interior, but no cavities were observed in the starch granules of non chalky endosperm in Y268F and R408G kernels due to that these starch granules were intact and not broken (Fig. 2B). In order to expose the inner structure of starch granules in Y268F and R408G kernels, the kernels were smoothly sectioned using a glass knife under ultramicro tome, and the starch granules were randomly cut to exhibit the micro structure of interior of starch granules. The cavities were clearly observed in the starch granules of nonchalky endosperm of GLXN, E410D, Y268F and R408G kernels (Fig. 2C). The size of cavity became smaller gradually from GLXN to E410D, Y268F and R408G (Fig. 2B, C, D). The linear fitting of AC and cavity area was analyzed among four rices (Fig. 2E). For four samples (n ¼ 4), the p is below 0.05 when the correlation coefficient (R) is over 0.950, and the p is below 0.01 when the R is over 0.990. In the present study, the R value ( 0.963) indicated that the area of cavity was significantly negatively correlated with the ACs (p < 0.05). The above results showed that starch granules with different ACs all had cavities in their interiors, but the starch granules with large cavities in glutinous and low AC rice were easily broken, and those with small cavities in high AC rice were difficultly broken.
3.3. Transparency of brown rice dry kernels with the same genetic background but different ACs The dry kernels of GLXN were waxy, E410D and Y268F presented opaque endosperms, and R408G had a transparent endosperm (Fig. 3A and B). Though the moisture of dry kernels showed no significant dif ferences among four rices (Fig. 3C), the transparency of kernels increased gradually from GLXN to E410D, Y268F and R408G (Fig. 3D). The transparency of dry kernel was significantly positively correlated with the AC (p < 0.05) (Fig. 3E) and negatively correlated with the cavity area of starch granules (p < 0.01) (Fig. 3F). Therefore, the transparency was influenced by the AC and cavity area of starch gran ules in rice endosperm. 3.4. Transparency and moisture of brown rice kernels with the same genetic background but different ACs during dehydration The freshly harvested brown rice kernels had high moisture. In order to clarify the effect of moisture on rice transparency, the freshly har vested brown rice kernels were oven-dried at 40 � C for different time. The freshly harvested kernels were transparent for GLXN, E410D, Y268F and R406G. During dehydration, the GLXN kernels gradually became waxy, the E410D, Y268F and R406G kernels gradually became opaque (Fig. 4). Though the transparency of kernels at the same dehydration time showed significant differences among four rice materials, they all
Fig. 4. Appearance of brown rice kernels from GLXN and its transgenic rice lines during dehydration at 40 � C. (A), kernels under reflected light; (B), kernels under transmitted light. Scale bar ¼ 1 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 5
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showed a decreasing trend with increase of dehydration time (Supple mental Table S1, Fig. 5A). The moisture of freshly harvested kernel was about 20%, and there were no significant differences among four rice materials. After dehydration for 48 h, the moisture of four lines was also similar (below 10%). However, the decreased rate of moisture was different among four rice lines during dehydration (Supplemental Table S2, Fig. 5B). The transparency was significantly positively corre lated with the moisture content for every rice material during dehy dration (p < 0.05 for GLXN, E410D and Y268F, and <0.01 for R408D) (Fig. 5C). Although there was a significant linear relationship between transparency and moisture of kernel, the slope constants (“K” values) of
linear fitting were significantly different among four rice materials (Fig. 5C). The “K” value represents the rate at which transparency changes with moisture content, and reflected the effect of moisture on transparency of kernel. The R408D line had significantly lowest “K” value (1.13), and the GLXN had the highest “K” value (2.14). The “K” value was significantly correlated negatively with AC (p < 0.05) and positively with the cavity area (p < 0.01) (Fig. 5D and E). These results showed that the transparency of kernel, especially for low AC rice, could be regulated through controlling the moisture of kernels, and the effect of moisture on transparency is influenced by AC and cavity size of starch granules.
Fig. 5. Transparency and moisture of brown rice kernels from GLXN and its transgenic rice lines during dehydration at 40 � C. (A), transparency; (B), moisture; (C), the relationship between transparency and moisture of brown rice kernels; (D), the relationship between “K” value and AC; (E), the relationship between “K” value and cavity area. The “K” value is the slope constant of linear fitting for transparency vs. moisture of kernels, and the “R” indicates the regression coefficient of linear fitting. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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4. Discussion
(Fig. 5D and E). In order to improve the appearance quality of kernel, soft rice on the market has high moisture in kernels (Lu et al., 2018). The present results showed that the transparency of kernels with low ACs could be regulated through controlling the moisture of kernels.
The rice transparency is an index for grading high-quality rice and has attracted a growing attention and interest. It is easier to observe and measure the transparency of kernels in polished rice than in brown rice. Therefore, the polished white rice, which is prepared through grinding the dry brown rice kernels with low moisture, is usually used to exhibit the transparency of rice kernels (Guo et al., 2011; Li et al., 2014, 2018; Lu et al., 2018; Wu et al., 2015). However, the freshly harvested brown rice kernels with high moisture are not suitable for preparing the pol ished rice. In fact, the transparency of brown rice can be detected and measured under both reflected and transmitted light, especially for the cross-section of kernel (Wu et al., 2001; Zeng et al., 2007). In order to investigate the relationship between transparency and moisture of rice kernels, we used the brown rice rather than the polished rice in the present study. Previous studies have described the decreased transparency as chalky, dull, milky white and opaque (Liu et al., 2009; Wan et al., 2005; Zhang et al., 2012). In this study, normal, soft and glutinous rice with different ACs were selected to further distinguish these phenotypes. All the three rice varieties contained chalky regions, but the chalky regions of soft and glutinous rice could be clearly seen only after rehydration of kernel (Fig. 1). The chalky regions of normal, soft and glutinous rices were packed loosely with starch granules having small size and spherical shape, and the nonchalky regions had polyhedral starch granules ar ranged closely in endosperm cells. Besides, many cavities were observed in the center of starch granules in the nonchalky regions of soft and glutinous rices (Fig. 1K and L). In conclusion, our results showed that chalkiness was not the cause for the decreased transparency of soft rice, and the cavities in the starch granules might be responsible for the poor transparency of dry kernels with low AC. Cavities have been observed in starch granules of soft rice Nanjing 46, Guandong 194 and Diantun 502 (Li et al., 2018; Lu et al., 2018). The starch granules of canna, potato and wheat also have cavities in their interior (Hall and Sayre, 1970, 1973; Baldwin et al., 1994). However, it is unclear what controls the size of cavity. In the present study, the glutinous rice GLXN and its derived three transgenic lines were selected to exclude the influence of different genetic backgrounds. Their starches have the same amylopectin structure, but significantly different ACs (Lin et al., 2018). The cavities could be observed in the broken starch gran ules of four rice materials, and their areas were significantly negatively correlated with the AC (Fig. 2). Normal starch granules are assembled by amylose and amylopectin. The amylose is concentrated mainly around the central hilum and the circumference of granule, and the amylopectin forms semicrystalline growth rings containing alternatively arranged crystalline and amorphous lamella (Cai et al., 2014; Glaring et al., 2006; Liu et al., 2019). The present results showed that AC was the critical factor determining the area of the cavity, and the lower the AC, the larger the cavity in the center of starch granules. Liu et al. (2009) have reported that three glutinous rice varieties with no AC show waxy phenotype and four Yunnan soft rice varieties with low AC (approximate 10%) show translucence. However, it is unclear for the relationship between AC and transparency of kernel. In the present study, linear regression analysis showed that the transparency was significantly positively correlated with the AC and negatively correlated with the area of cavity (Fig. 3E and F). Li et al. (2018) have found that the polished rice with 10.6% AC requires less time than the normal rice and more time than the glutinous rice to change from transparent to opaque during kernel drying. In the present study, the freshly harvested brown rice kernels changed from transparent to opaque in E410D, Y268F and R408G lines or waxy in GLXN during kernel dehydration (Fig. 4). Linear regression analysis showed that the transparency of kernel was positively correlated with moisture during dehydration, and the rate “K” value of transparency vs. moisture is different among four rice materials (Fig. 5C). Meanwhile, the “K” value was significantly correlated negatively with AC and positively with the cavity area
5. Conclusion Starch granules in brown rice dry kernels had cavities in their in teriors. The cavity size was negatively correlated with AC. A poor transparency of dry kernels with low AC was caused by the cavities of starch granules. The transparency of dry kernels with similar moisture was significantly correlated positively with AC and negatively with cavity size of starch granule. For kernels from the same rice variety, the transparency was significantly positively correlated with the moisture of kernels. The effect of moisture on transparency of kernel was correlated negatively with AC of kernel and positively with cavity size of starch granule. The transparency of low AC rice could be regulated through controlling the moisture of kernels. Declaration of competing interest The authors declare no conflict of interest. Acknowledgements This study was supported by grants from the Natural Science Foun dation of Jiangsu Province (BK20160461), the National Natural Science Foundation of China (31901427), the China Postdoctoral Science Foundation (2018T110561), the Talent Project of Yangzhou University, and the Priority Academic Program Development of Jiangsu Higher Education Institutions. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.jcs.2019.102854. References Baldwin, P.M., Adler, J., Davies, M.C., Melia, C.D., 1994. Holes in starch granules: confocal, SEM and light microscopy studies of starch granule structure. Starch 46, 341–346. Cai, C., Huang, J., Zhao, L., Liu, Q., Zhang, C., Wei, C., 2014. Heterogeneous structure and spatial distribution in endosperm of high-amylose rice starch granules with different morphologies. J. Agric. Food Chem. 62, 10143–10152. Chen, L., Gao, W., Chen, S., Wang, L., Zou, J., Liu, Y., Wang, H., Chen, Z., Guo, T., 2016. High-resolution QTL mapping for grain appearance traits and co-localization of chalkiness-associated differentially expressed candidate genes in rice. Rice 9, 48. Fitzgerald, M.A., McCouch, S.R., Hall, R.D., 2009. Not just a grain of rice: the quest for quality. Trends Plant Sci. 14, 133–139. Glaring, M.A., Koch, C.B., Blennow, A., 2006. Genotype-specific spatial distribution of starch molecules in the starch granule: a combined CLSM and SEM approach. Biomacromolecules 7, 2310–2320. Guo, T., Liu, X., Wan, X., Weng, J., Liu, S., Liu, X., Chen, M., Li, J., Su, N., Wu, F., Cheng, Z., Guo, X., Lei, C., Wang, J., Jiang, L., Wan, J., 2011. Identification of a stable quantitative trait locus for percentage grains with white chalkiness in rice (Oryza sativa). J. Integr. Plant Biol. 53, 598–607. Hall, D.M., Sayre, J.G., 1970. Internal architecture of potato and canna starch: Part I: crushing studies. Text. Res. J. 40, 147–157. Hall, D.M., Sayre, J.G., 1973. A comparision of starch granules as seen by both scanning electron and ordinary light microscopy. Starch 25, 119–123. Li, Q., Huang, L., Chu, R., Li, J., Jiang, M., Zhang, C., Fan, X., Yu, H., Gu, M., Liu, Q., 2018. Down-regulation of SSSII-2 gene expression results in novel low-amylose rice with soft, transparent grains. J. Agric. Food Chem. 66, 9750–9760. Li, Y., Fan, C., Xing, Y., Yun, P., Luo, L., Yan, B., Peng, B., Xie, W., Wang, G., Li, X., Xiao, J., Xu, C., He, Y., 2014. Chalk 5 encodes a vacuolar Hþ-translocating pyrophosphatase influencing grain chalkiness in rice. Nat. Genet. 46, 398–404. Lin, L., Zhang, L., Cai, X., Liu, Q., Zhang, C., Wei, C., 2018. The relationship between enzyme hydrolysis and the components of rice starches with the same genetic background and amylopectin structure but different amylose contents. Food Hydrocolloids 84, 406–413. Liu, D., Wang, W., Cai, X., 2015. Modulation of amylose content by structure-based modification of OsGBSS1 activity in rice (Oryza sativa L.). Plant Biotechnol. J. 12, 1297–1307.
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