Scanning Electron Microscopic Observation on Chalkiness of Rice Mutant O s LHT1 Grains

Grain chalkiness greatly affects the grain appearance ， milling ， eating ， cooking ， and nutritional qualities ， so it is one of the most important traits of grain qualities. To study the relationship between chalkiness of mutant of rice OsLHT1 gene and the shape, structure and arrangement of endosperm cells and starch grains, the chalkiness rate, chalkiness degree and chalkiness area of the mutant of rice OsLHT1 gene were investigated by field experiment, and the morphological structure of rice endosperm cells and starch grains were also observed by scanning electron microscope. Our results showed that the grain chalkiness character with the greatest difference between the tested mutant of OsLHT1 gene and the wild japonica rice variety Zhonghua 11 is chalkiness degree, followed by chalkiness rate, and finally chalkiness area, and there is a significant correlation between chalkiness rate and chalkiness degree. Therefore, there is a close correlation between the arrangement of endosperm cells, the distribution of starch grains and the occurrence of grain chalkiness in the mutant of OsLHT1 in japonica rice.


Introduction
Rice (Oryza sativa L.) is one of the most important food crops in the world. More than half of the world's population and two thirds of China's population take rice as their staple food (Chen et al., 2019). For a long time, China's rice breeding has taken yield as the main breeding goal, and paid less attention to rice quality. However, with the improvement of people's living standards, the demand for high-quality rice is increasing. Therefore, cultivating new rice varieties with high quality has become one of the important objectives of current breeding work. The quality of rice mainly involves processing quality, appearance quality, cooking and eating quality and nutritional quality. Processing quality can be reflected by such indicators as brown rice rate, milled rice rate and whole milled rice rate Ding et al., 2020;Wang et al., 2018). The cooking and eating quality and nutritional quality of rice are closely related to the protein content of rice, and the cooking and eating quality determines the acceptance of rice by consumers (Zhang et al., 2017). The appearance quality is an important character index to measure the commercial value of rice, which is mainly reflected by rice grain size, length width ratio, transparency, chalkiness rate and chalkiness (Zhang et al., 2012;. Chalkiness not only affects the appearance quality of rice, but also affects the grinding and processing quality, nutritional quality, cooking and eating quality of rice, and even leads to the reduction of the final yield of rice (Peng et al., 2019). Therefore, chalkiness directly determines the quality of rice and the market price to a large extent.
Chalkiness is one of the most important appearance quality traits, and also one of the key factors affecting the cooking and eating quality of rice . Chalkiness refers to the loose arrangement of starch and protein particles in rice endosperm due to the rapid filling rate, and the formation of a large number of voids after drying and water loss, resulting in light scattering and the formation of white opaque parts, including abdominal white, back white and heart white. It is the quality index most affected by the environment (Chen et al., 2013;Li et al., 2014). Rice with chalkiness has poor transparency, low head rice rate and poor commodity quality [13] . Reducing chalkiness is a key problem to be solved in rice quality improvement. Different varieties and qualities of rice have their fixed starch grain morphology, and the quality of rice can be preliminarily determined by scanning electron microscopy (Peng et al., 2014), thus providing a basis for identification for breeding high-quality rice.
Studies have shown that rice chalkiness is a quantitative trait jointly controlled by multiple genes (Qiu et al., 2014)., with additive effect and interaction effect between additive effect and environment, and affected by external environmental factors (Lv et al., 2021;Peng et al., 2021). For example, high temperature induction, daily average temperature, temperature difference between day and night, sunshine duration, cultivation measures and other factors in the filling period will have different degrees of influence on the chalkiness character of rice (Bai et al., 2021;Zheng et al., 2022;Yan et al., 2021;Abayawickrama et al., 2017). At present, a large number of genes affecting rice chalkiness have been isolated and cloned by using natural populations or mutants, such as gw2, chalk5, osppdkb, ssiiia, gif1, osrab5a, FLO2, osssi, flo4, flo15, etc. (Bahuguna et al., 2017;Deng et al., 2021;Deng et al., 2018;Lu et al., 2018;Sheng et al., 2015;Zhu et al., 2020;Ying et al., 2019). It can be roughly divided into three categories: one is the main gene for rice chalkiness, and currently only chalk5 gene (Guo et al., 2020). One is that genes controlling other traits can also affect chalkiness. Such genes are usually expressed as one cause and multiple effects, such as gw2 gene controlling grain width (Tsuyoshi et al., 2017). Another is that genes involved in starch synthesis, such as FLO2, flo4, flo15, etc., can also affect chalkiness Zhou et al., 2015;Luo et al., 2021;Tabassum et al., 2020). Previous studies have shown that OsLHT1 gene is a protein gene regulating histidine transport in rice. It is expressed in important growth stages of rice, and the expression level is highest in roots and old leaves at tillering stage. Loss of function of OsLHT1 gene will inhibit the growth of rice roots and stems, leaves will appear obvious brown spots, plants will show signs of premature senescence, and the 1000 seed weight and seed setting rate will be significantly reduced (Zhu et al., 2018;Li et al., 2017;Hu et al., 2019). However, it is not clear whether OsLHT1 gene affects rice quality traits, especially chalkiness. Therefore, in this study, rice OsLHT1 mutants were used as experimental materials to detect and analyze the Chalkiness Characters in their grains, providing reference for further study on the function of OsLHT1 gene and its potential application in the breeding of new rice varieties.

Test Materials
The tested material was rice mutant OsLHT1, and the corresponding parent material was Zhonghua 11 (zh11). The rice mutants were derived from the rice mutant library of Huazhong Agricultural University.

Field Planting
In the summer of 2021, the mutant OsLHT1 plants and zh11 were planted in the same experimental field of Xinyang Normal University. The mutants OsLHT1 and zh11 were planted in two rows, 12 plants in each row, and the plant row spacing was 16.5 cm × 26.4 cm. From sowing to seed maturity, the tested materials were managed by conventional cultivation in the field. After the seeds were mature, they were naturally dried and stored at room temperature for 3 months, and then the chalkiness characters were tested.

Chalkiness Character Determination
The chalkiness rate, chalkiness area and chalkiness degree of Japonica rice varieties were determined and analyzed according to the national standard GB/t17891-1999. Take 100 full and complete milled rice randomly from the mutant OsLHT1 and zh11, count the chalky rice grains, repeat for 3 times, and take the average value as the chalkiness rate. The belly white rate, the heart white rate and the back white rate were measured according to the method similar to the chalkiness rate, that is, 100 full and complete milled rice were randomly taken, and the rice grains with belly white and heart white were counted, and repeated three times. The average value was taken as the belly white rate and the heart white rate. Similarly, 10 chalky rice grains were randomly selected from the mutant OsLHT1 and zh11, and the percentage of chalky rice grains in the whole rice grain area was estimated.

Identif
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Differe
According belly whit and back o the mutant with good mutant Os abdomen a rice OsLH grains in th starch grai   Vol. 14, No. 11; have shown that if a single starch grain can achieve a composite starch grain with a certain geometric shape and orderly arrangement, chalkiness will not appear in this part [38][39] . Ellipsoidal or spherical starch grains cannot be arranged orderly in the chalky part, and the deposition is disordered. The deposition of such ordered and disordered starch grains is reflected in the appearance, that is, whether the rice endosperm is chalky and whether it is transparent. However, after the mutation of rice OsLHT1 gene, the gap of starch grains increases, the arrangement is loose, and the development of starch grains is poor. Therefore, chalkiness characters are easy to be produced, and the chalkiness rate and chalkiness degree of the corresponding positions are also increased, which further indicates that there is a close relationship between the distribution of starch grains on the cross section of rice endosperm and the production of Chalkiness Characters.
By observing the chalkiness of the endosperm of the mutant OsLHT1, we found that after the mutation of the rice OsLHT1 gene, the arrangement of the endosperm cells also changed from the central part to the surrounding, and the shape of the endosperm cells was mostly spherical, with different sizes. The ventral White was significantly increased, and most of the endosperm cells were arranged in this form, indicating that the arrangement of the endosperm cells had a certain correlation with the generation of the chalkiness of rice. Combined with our previous research results, it showed that the chalkiness of endosperm was mainly controlled by genetics. However, the occurrence of chalkiness is a complex process, and the formation of chalkiness may involve the relationship between "source", "sink" and "flow" in rice plants. Any change in any one of the three or the disharmony of their relationship may lead to or increase the formation of Chalkiness in rice endosperm (Jiang et al., 2018;Nevame et al., 2018;Huang et al., 2020).
According to the results of this study, we believe that the chalkiness degree, chalkiness rate and chalkiness area of rice increased significantly after the mutation of OsLHT1 gene, and the increase of chalkiness degree and chalkiness area reached a very significant level. In the grain of the japonica rice mutant OsLHT1, the arrangement of endosperm cells showed a tendency of radiating from the center to the periphery, and the shape was mostly spherical, with different sizes. The distribution of starch grains is loose and irregular, thus forming a high chalkiness character. The results showed that the structure of endosperm cells and cross-sectional starch grains determined the appearance of chalkiness, and the endosperm structure was determined by the development of rice endosperm cells, all of which were controlled by genetic factors. Therefore, the occurrence of Chalkiness in rice endosperm is closely related to the distribution of starch grains in a certain position and the development of starch grains in that position. Thus, it will lay a foundation for studying the mechanism of chalkiness formation and cultivating high quality rice varieties in the future.

Conclusion
Chalkiness is one of the most important appearance quality traits, and also one of the key factors affecting the cooking and eating quality of rice. The chalkiness of rice is not only affected by environment, but also by genetic factors. In recent years, the development of rice functional genome has greatly promoted the research on the genetic mechanism of chalkiness and the mining of favorable genes. However, at present, the mechanism of Chalkiness of mutant OsLHT1 and the relationship between gene and chalkiness are not clear. Through this experiment, it was found that the chalkiness rate, chalkiness degree and chalkiness area of rice OsLHT1 gene were significantly higher than that of wild-type zh11, and the arrangement of starch grains was loose and irregular, and the starch development was also poor. Therefore, it can be speculated that OsLHT1 gene mutation may lead to Chalkiness in rice. It also showed that the formation of Chalkiness in rice was closely related to the shape structure and arrangement of starch grains, so it could be used for the identification of rice quality.