The Arrangement of Endosperm Cells and Development of Starch Granules Are Associated With the Occurrence of Grain Chalkiness in Japonica Varieties

The endosperm chalkiness in grains is one of the most important traits in rice grain qualities, and grain chalkiness directly determines grain quality and market price. Therefore, the endosperm chalkiness reduction is an important goal of breeding scientist in rice. However, little is known about the relationship of the formation of rice endosperm chalkiness and the morphological structure and development in fragrant japonica and conventional japonica varieties. In this study, we have mainly investigated the chalkiness characters such as degree of endosperm chalkiness (DEC), chalkiness rate (CR) and chalkiness area (CA) in 12 fragrant japonica varieties and 9 conventional japonica varieties. Furthermore, the endosperm structure and starch granules of rice grains were also observed with scanning electron microscope (SEM). The results indicated that the 21 varieties including the different fragrant japonica and conventional japonica varieties has a linear relationship between the DEC and the CR (P < 0.05). Among the fragrant japonica and conventional japonica varieties, the biggest difference is the CR, follow by CA and DEC. In addition, there is a certain correlation between the arrangement of endosperm cells, the distribution of starch granules and the occurrence of chalkiness characters in the different fragrant japonica and conventional japonica varieties. For the same fragrant japonica or conventional japonica variety, there is no significant difference between the starch granules from the transparent parts of chalkiness and non-chalky, while the starch granules of grains between chalky and non-chalky has obvious difference. Our results would provide important references for high quality rice breeding.


Introduction
Rice (Oryza sativa L.) is one of the most important food crops in the world, and the staple food for more than half of the world's population (Balindong et al., 2018;Zhao et al., 2013).Along with the sustained development of living standards, people's demand for high quality rice is increasing (Zhou et al., 2015;Fitzgerald et al., 2009;Cheng et al., 2007).However, rice grain quality is a complex quantitative trait which consists of milling, appearance, eating, nutritional, cooking and eating qualities (Tian et al., 2009;Peng et al., 2014;Liu et al., 2010).Among these qualities, consumers often focus on appearance quality and cooking and eating quality (Guo et al., 2011;Cuevas et al., 2016).The appearance quality of rice grain, including grain length, grain width, grain thickness and chalkiness or transparency (Qiu et al., 2015), though, grain chalkiness not only affect the rice appearance quality but also influence the milling, cooking and eating qualities (Liu et al., 2010;Guo et al., 2011;Sun et al., 2015;Shen et al., 2007;Yoshioka et al., 2007).Meanwhile, chalkiness characters will reduce head milled rate and thus affect its production in grain, and has a great impact on the price of rice (Cuevas et al., 2016;Sun et al., 2015;Zhao et al., 2013).Therefore, grain chalkiness character is the main determinant of grain qualities and market value of rice.
Chalkiness is one of the main problems in rice production and consumption; there are severe problems of high chalkiness rate and chalkiness degree in many rice varieties.Therefore, reducing the grain chalkiness is an important goal of rice breeders and botanist (Guo et al., 2011;Qiu et al., 2015).Previous studies have indicated that grain chalkiness is a complex character and polygenic controlled quantitative trait loci (QTL) (Peng et al., 2014;Qiu et al., 2015;Liu et al., 2010;Peng et al., 2016)，and that there are additive effect and interaction effect between the additive effect and environments (Yoshioka et al., 2007;Li et al., 2014;Tsukaguchi et al., 2008;Peng et al., 2004;Mei et al., 2013;Yamakawa et al., 2008;Wan et al., 2005).At present, more than 140 QTLs controlling grain chalkiness characters have been detected using different type of populations, and these QTLs were distributed on the 12 chromosomes of rice, but, only a few QTLs controlling the trait were obtained for isolation and cloning and function analysis (Peng et al., 2014;Peng et al., 2016;Lin et al., 2016).Some QTLs and genes affecting chalkiness characters were isolated and cloned by natural populations or mutants, i.e.GL7 (Wang et al., 2015), Chalk5 (Li et al., 2014), GW2 (Song et al., 2007), OsPPDKB (Kang et al., 2005), SSIIIa (Fujita et al., 2007;Ryoo et al., 2007), GIF1 (Wang et al., 2010a), OsRab5a (Wang et al., 2010b), flo2 (She et al., 2010).The slow progress in the study of chalkiness partially reflects the complexity of the mechanism underlying grain chalkiness occurrence, which needs intensive studies (Lin et al., 2016).In addition, there is a variety of factors affecting the formation of the grain chalkiness (Yu et al., 2015;Zhao et al., 2015;Zhou et al., 2015), such as starch synthesis, starch granule structure and arrangement, and external environmental factors including high temperature in grain filling period, daily mean temperature, temperature difference of day and night, sunshine duration and cultivation measures.
Compared with non-chalky rice, the chalky grains have the following characteristics, amylose and long branched chain starch contents decreased significantly, short branched chain starch content was significantly increased and head rice percentage and transparency was reduced, consequently, grain eating quality and yield will be debased (Cuevas et al., 2016;Qiu et al., 2015;Patindol et al., 2003).Chalkiness is a white and opaque part in grain, the arrangement of starch granules is loose and inflated, which is result from the influence of development and enrichment of endosperm starch granules and proteoplast at pustulation period (Zhou et al., 2015;Patindol et al., 2003;Li et al., 2014).There are consistent starch granule structure in all rice varieties, therefore, we can preliminary identify the advantages and disadvantages of rice quality using scanning electron microscopy (SEM) (Lisle et al., 2000;Fitzgerald et al., 2009).Most japonica varieties have excellent grain quality, endosperm chalkiness characters are remarkable differences among different rice varieties.Fragrant japonica is a special type of japonica rice and has the higher price due to its fragrance and high nutrition value, which are consider as treasures in rice (Kovach et al., 2009;Daygon et al., 2016;Shao et al., 2013).Previous studies showed that the heritability of grain chalkiness characters is relatively lower in fragrant japonica, and found that chalkiness characters are related to the content and structure of starch (Sun et al., 2015;Zhou et al., 2015;Chakraborty et al., 2016).However, there is still no breakthrough in the progress of mechanism study of grain chalkiness characters formation in japonica varieties especially in fragrant japonica.
In this study, we have mainly investigated the CR, DEC and CA of 12 different fragrant japonica and 9 conventional japonica varieties, and rice endosperm cells and starch granules were observed using SEM.Moreover, the correlation between the formation of chalkiness characters, morphology structure and development of endosperm cells and starch granules were also studied in the 21 rice varieties.Thus, our results will provide useful references for the formation mechanism of grain chalkiness characters in fragrant japonica and conventional japonica varieties, and the grain quality improvement in rice breeding.

Materials
All tested rice varieties are japonica varieties, including 12 fragrant japonica and 9 conventional japonica varieties, and most of them come from China.There are 7 fragrant japonica varieties (Xinxianggeng 1, Xiaobao 1, Xianggeng 1862, Xiangbao 2, Xianggeng 33, Nongxianggeng, Nongxianggeng 4) and 3 conventional japonica varieties (Zhengdao 02, Zhengdao 18, Xingengnuo 631) from Henan province, 3 fragrant japonica varieties (Xianggeng 805, Wuxianggeng 23, Guanglingxianggeng) and 1 conventional japonica variety (Longgeng 31) from Jiangsu province, 1 fragrant japonica variety (Changxianggeng 101) and 1 conventional japonica variety (Longgeng 31) from Heilongjiang province, 1 conventional japonica variety (Liaoxing 1) from Liaoning province, 1 conventional japonica variety (Xiushui 134) from Zhejiang province, 1 conventional japonica variety (Yilatai 104) from France and 1 conventional japonica variety (Huangjinqing) from Japan.According to the different growth stage of these rice varieties, and to ensure that the 15 japonica varieties which blossom in late August and experience the relatively consistent temperature of grain filling period, these varieties were sowed in batches in a same experiment field of Xinyang City Academy of Agricultural Sciences in 2016 and 2017.Each line was planted with 2 replications in each sowing, with each line containing 12 plants transplanted in a single row with 16.5-cm plant spacing and 26.4-cm row spacing.Field management essentially followed normal agricultural practice.Field irrigation was maintained to avoid drought stress to the late-maturing lines (Peng et al., 2014;Fan et al., 2005), matured rice grains were tested after air drying and room temperature for three months.

Determination of Chalkiness Characters
CR, CA and DEC were measured and analyzed with the National Standard of the People's Republic of China-Good Quality of Rice grains (GB/T17891-1999).In each variety 100 full and complete milled grains were randomly selected, then counted the numbers of grains with chalkiness (repeated 3 times), and the average value was taken as the CR of each variety; the method of measuring white belly rate and white core rate is similar to the method of CR, i.e. randomly select 100 full and complete milled grains, count the numbers of grains with white belly and white-cored and repeated 3 times, respectively, and the average value is taken as the white belly rate and white core rate; To estimate the percentage of the chalkiness area in whole area of grain, 10 grains with chalkiness were selected randomly in different varieties (repeated 3 times), and the average area is taken as the CA (Peng et al., 2014).DEC is equal to CR multiply by CA [DEC = CR × CA].SPSS software was used to analyze the variance of the measured data.

Scanning Electron Microscope Observation
Randomly we selected the grains with chalky and non-chalky in each varieties, for scanning electron microscopy, and milled rice grains were transversely cut at the middle with a knife and coated with gold under HUS-5GB vacuum evaporation equipment.The morphology of starch granules in different part of the endosperm was examined by a scanning electron microscope (Hitachi S-4800).The SEM analysis was based on at least three biological replications of the mounted specimens.All procedures were according to the manufacturer's protocol (Li et al., 2014).

Difference of Chalkiness Characters in Different Fragrant Japonica and Conventional Japonica Varieties
The results of chalkiness characters in the 12 fragrant japonica varieties and the 9 conventional japonica varieties are shown in Figure 1.Among these japonica varieties, the biggest differences were observed for the CR, follow by CA and DEC.There are higher CR with more than 98% in Xingengnuo 631, Heixiangdao 193 and Xiangbao 2, while basically no chalkiness characters were observed in Huangjinqing, Nongxianggeng 4 and Longgeng31.The CR ranged from 2.9% to 100% in the 21 japonica varieties.Variance analysis showed no significant difference of CR among Xinxianggeng 1, Xiangbao 1, Changxianggeng 101 and Wuyungeng 23; also, no significant difference of CR among Xinggengbuo 631, Heixiangdao 193 and Xiangbao 2; likewise, no significant difference of CR among Huangjinqing, Nongxianggeng 4, Longgeng 31 and Xiushui 134; and no significant difference of CR among Nongxianggeng and Yilatai 104.Change trend of DEC was quite similar to the CR in the 21 japonica varieties, and the DEC ranged between 0.3% and 100%.
The chalkiness of Changxianggeng 101, Nongxianggeng, Xianggeng 805, Guanglingxianggeng, Zhengdao 02, Liaoxing 1 and Wuyungeng 23 is mainly occurred on the belly of grains, and that is called white belly; the chalkiness characters of Xianggeng 1862, Xiangbao 2, Xianggeng 33 and Yilatai 104 is mainly occurred in center of rice, known as white core.In addition, there are both white belly and white core in Xinxianggeng, Xiangbao 1 and Wuyungeng 23 varieties.However, other varieties' CA was very low and even no-chalky present in grains.The range of CA is from 2.2% to 100%, and the change trend is different to CR and DEC.The CR of Xinxianggeng 1, Changxianggeng 101 and Xianggeng 805 are all more than 27.0%, but the CA are below 7.0%.Interestingly, the chalkiness characters are almost not observed in Huangjinqing, while the CR is up to 98.4%, CA up to 51.8%, and DEC up to 51% in Xiangbao 2. Further analysis shows that the linear relationship between the CR an CR is often Note.CR,

Morph Varieties
From

Grain Chalkiness and Development of Starch Granules in Fragrant Japonica and Conventional Japonica Varieties
In this study, the observation of chalkiness characters in the 12 fragrant japonica varieties and the 9 conventional japonica varieties, Showed that the morphological structure and arrangement of starch granules in grain endosperm cells were different in the different parts of the same japonica varieties.Compared with the starch granules from the grains of transparent parts or without grain chalkiness varieties, the starch granules were mainly in the free state of existence and cannot further developed into certain form of starch granules complex in fragrant japonica and conventional japonica varieties.So, if the individual starch granules can be closely ordered into some form of starch granules complex, the grain chalkiness characters would not occurre at this site.Though, if the spherical or ellipsoidal starch granules is arranged irregularly in the grain chalkiness regions, the deposition showed disordered state and the deposition of starch granules are reflected in the appearance of the grains, which means that the grain endosperm has chalkiness characters and its performance is not the transparent state.In addition, previous studies have shown that the development in the back of endosperm cells is relatively better than that in the center and belly of grain endosperm, which showed so little or no white back in grains (Li et al., 2014;Sing et al., 2003;Kim et al., 2004).We also found no white back in grains, and starch granules of back are in the rules of the diamond or other polygons with polyhedral arris, dense arrangement, small and even no gap between starch granules and the starch granules of back developed well in grains.When testing endosperm of 12 fragrant japonica and 9 conventional japonica varieties in this study, the reason could be that nutrients are first transported to the back of the japonica rice seeds, and then to the central part of the endosperm and the abdomen, this makes the development of back of the grain endosperm relatively good.Therefore, we may say that there is close relationship between grain chalkiness characters of endosperm and development of starch granules in fragrant japonica and conventional japonica varieties.

Reasons for the Formation of Grain Chalkiness
Previous studies have shown that grain chalkiness is a complex quantitative trait, which is controlled by multiple genes and readily influenced by environmental factors (Qiu et al., 2015;Guo et al., 2011;Li et al., 2014;Tsukaguchi et al., 2008;Peng et al., 2014;Peng et al., 2016).In this study, among the 12 fragrant japonica and the 9 conventional japonica varieties, there were obvious differences in chalkiness characters in grain endosperm, and if we combined it with our preliminary findings, we can concurre that chalkiness characters of grain endosperm are mainly controlled by genes.However, it is also known that the occurrence of grain chalkiness is a very complex process, in which the relationship between the "source", "sink" and "flow" may be involved in the formation of endosperm chalkiness (Lin et al., 2016;Kim et al., 2004).Thus, a change or uncoordinated in any of these three relations are likely to cause or increase the opportunity of the formation of endosperm chalkiness characters.In regard of what preceded, we cool consider that endosperm cells and the structure of starch granules on the cross section determine grain chalkiness characters, and endosperm structure is influenced by the development of rice endosperm cells, which are all controlled by genetic factors.Therfore, the happening of grain chalkiness in fragrant japonica and conventional japonica varieties is intimately related to the distribution and development of starch granules in certain region of grain endosperm, and it could be very critical for the occurrence, development, later deposition patterns and distribution ways of starch granules in rice endosperm.However, little is known about the starch granules of early occurrence, development and later deposition pattern in fragrant japonica and conventional japonica varieties.Indeed, many questions still unanswered up to now, such us, how many genes might be involved in this important regulation process?The interaction of these genes?How is played the key role for starch granules in the process of chalkiness formation in fragrant japonica and conventional japonica varieties?The solution of these crucial issues) will provide important information for further clarifying the mechanism of the occurrence of grain chalkiness characters in rice.
Of all what preceed, the present study found that both the arrangement of endosperm cells and distribution of starch granules are associated with the occurrence of grain chalkiness in fragrant japonica and conventional japonica varieties.Therefore, we can easily say that our results could help to pave a new way for the improvement of grain chalkiness and grain quality in japonica varieties and, potentially, other staple crops.