4.1 Formation characteristics of rice endosperm structures
Rice quality is closely related to endosperm structures 8, 14-23. Previous studies on endosperm structures mostly focused on the analysis of rice quality and traits in the mature stage, but the dynamic changes of morphological characteristics during the formation of rice quality were seldom explored. In this study, the dynamic changes of starch accumulation and endosperm structure formation during the rice quality formation process of two rice cultivars of three different genotypes were explored. The microstructure formation process of rice endosperm was displayed from the the perspectives of morphology and anatomy. It is significant for understanding the internal mechanism of rice quality formation and improving breeding and cultivation of high-quality rice.
During the filling process, the morphological changes of the endosperm structure of grains included the increased quantity and size of amyloplasts, the decreased gaps, and the tighter arrangement. In this study, starch accumulation was observed in grains in 5 DAF. The formation of endosperm structures in different grain positions was significantly different. The formation of endosperm amyloplasts in early-flowering grains was significantly earlier than that of late-flowering grains and amyloplasts gaps in early-flowering grains were also significantly smaller than those in late-flowering grains. Amyloplasts gaps were distributed according to the flowering order of spikelets.
In this study, in the grains at the same grain position in the same filling period, the formation of endosperm structures in glutinous rice was the earliest, followed by indica rice and the formation of endosperm structures in japonica rice was the latest. This differences in the formation of endosperm structure between different genotypes could be observed even in 25 DAF. This was consistent with the previous reports that the inferior grains of japonica rice had almost no dry matter accumulation in 5-10 DAF 24.
4.2 Relationship between endosperm structural characteristics and plumpness
The endosperm amyloplasts were characterized by the tight arrangement, small gaps and high plumpness 21. The plumpness data of rice cultivars of different genotypes (Table 1) showed that the plumpness of inferior grains of each cultivar was lower than that of superior grains. Among various rice cultivars, glutinous rice had the best plumpness, followed by japonica rice, and indica rice Yangdao 6 had the lowest plumpness. The endosperm structure of late-flowering grains in the middle panicles of Yangdao 6 in the mature stage was slightly looser. The endosperm structure of glutinous rice was tight, indicating that the difference in endosperm structure between different rice genotypes in the mature stage was consistent with the plumpness difference (Table 1).
In this study, the formation of endosperm structure was different in different position of grains. The endosperm structure of mature rice was better in the back than that in the center and the belly. In 5-10 DAF, the endosperm in the center was fuller than that in the back and belly. The results were different from the previous studies on the endosperm structure and did not seem to conform to the principle of nearby transportation of materials 8,12-19 The differences might be interpreted as follows. Firstly, the photosynthetic product was transported to grains in the form of sucrose through phloem and the long transport route of filling materials determined that there was a sucrose concentration gradient inside grains 25. Secondly, the sucrose concentration in the center was the lowest, thus more sucrose from the surrounding accumulated in the center. Thirdly, due to the small size of center cells, the sucrose concentration required for starch synthesis was reached more earlier to form amyloplasts. Fourthly, from the perspective of biological adaptability, the first formation of starch granules in the center was beneficial to the filling process of the entire grain. Otherwise, the formation of starch granules in the back or periphery would increase the filling resistance of materials into the inner endosperm cells, thus increasing the difficulty in filling the center of grains. Perhaps this is exactly the result obtained after the long evolution process of rice. Generally speaking, in 20 DAF, amyloplasts in the center of superior grains was tighter, but the size was small.
In 30-35 DAF-the mature stage, the differences among different position of the endosperm structure of a grain were further reduced. According to the principle of material transportation nearby, the back of the grain was filled before the belly. In 30-35 DAF-the mature stage, early-flowering grains were mainly filled in the belly, whereas late-flowering grains were filled in the core and belly as well as the back.
In 30-35 DAF-the mature stage, the differences among different grain location on a panicle were further reduced until the original filling rule was changed. Late-flowering grains had a slow filling rate and a long filling period. Inferior grains were still actively filled in 20-30 DAF, and starch accumulation in these grains could be always observed in 30-40 DAF. Therefore, in the late-flowering grains on the primary or secondary branches, starch accumulation or filling rate was not necessarily the worst, indicating that the filling trend of late-flowering grains could be reversed. This filling feature suggested that the cultivation and management of rice during the entire filling period was the focus. Therefore, the appropriate application of spikelet fertilizer, irrigation in the grain filling period, and delayed suspension of water to prolong the starch accumulation which might be conducive to the filling process of rice and the overall quality of rice.
4.3 Relationship between endosperm structural characteristics and grain transparency
Careful observations under a scanning electron microscope indicated that glutinous rice had three types of pores: pores in starch granules, gaps between single starch granules in amyloplasts, and micropores in membranous structures (from membrane or cell wall). The three types of pores did not appear in the early filling stage, but they appeared in the middle and late filling stages. According to microscopic observations, the surface of the early amyloplasts of glutinous rice was the roughest and the membrane was the thickest. We reasonably believed that the thick membrane concealed the holes that originally existed in starch granules until these holes appeared due to the dehydration and drying process in the later stage. According to our analysis, all rice cultivars experienced a process of water loss during the grain filling stage and the water loss process was not limited to glutinous rice. The endosperm of glutinous rice contained amylopectin and little amylose. The Wx gene of glutinous rice determined this trait. Amylose molecules were located in the center of starch granules, whereas amylopectin was located in the periphery of starch granules (Nielsen et al., 2002). Therefore, the starch granules of glutinous rice were different from those of indica and japonica rice at the beginning of formation. The holes in starch granules were not visible in the early stage of filling due to the thick membrane and the high water content. In the middle and late stages, especially in the mature stage, a large amount of the holes in starch granules were exposed due to the gradual water loss during the mature transition stage. It should be pointed out that in the two tested cultivars of indica rice and japonica rice, pores were also observed. The pores in indica and japonica rice were micropores of membranous structures (membrane or cell wall). These micropores were also observed in glutinous rice. Therefore, the micropores on this membrane structure were the common feature of different rice cultivars and the diameter of micropores was small and shallow. Compared with the other two forms of pores in glutinous rice (the holes on starch granules with the diameter of 0.7-1.34 mm), the gaps between starch granules in amyloplasts (3.98 mm × 1.14 mm) were different in position, quantity and size.
If the degree of plumpness was good, the amyloplasts in endosperm were tightly arranged and the endosperm appearance was transparent. When the plumpness was poor, the amyloplasts in endosperm were loosely arranged and the endosperm appearance was not transparent. However, the opacity of glutinous rice did not mean that the amyloplasts in endosperm was poorly filled. The opaque appearance of glutinous rice was caused by too many voids in the endosperm and air refraction. The amyloplasts in endosperm of glutinous rice were also loosely arranged and poorly filled, but the phenomenon mainly occurred in the belly and center of inferior grains. Therefore, the space caused by poorly filled amyloplasts in the endosperm were the secondary structural characteristics responsible for the opaque appearance of glutinous rice. In the mature endosperm of glutinous rice, amyloplasts contained a large number of pores, as previously reported 13,27. The pores in the starch granules and the obvious gaps between starch granules were two types of unique pores of glutinous rice and responsible for the opaque appearance of glutinous rice.
In scanning electron microscope images, under conventional cultivation conditions, the microporous structures with a diameter of about 0.2 mm appeared on starch granules in the belly of the 2nd grain on the primary branch of Huajing 2 and in the center of the 4th grain on the secondary branch of Yangdao 6. The microporous structures were most likely to occur in the belly of endosperm adjacent to the embryo because even in the seed storage period, the embryo breathed and consumed energy. The nearby belly of endosperm provided energy, so that the nearby amyloplasts were consumed and decomposed. Thus many small holes were formed in the endosperm. The phenomenon was also common in the process of seed germination. Under weak light, high temperature, and low-temperature stress, holes were formed in starch granules 15,28. Interestingly, under conventional cultivation conditions, in the belly of late-flowering grains on the secondary branches of the middle and lower panicles of conventional rice (Fig. 5(E3)), the phenomenon often appeared in 20 DAF, it indicated a certain obstacle to amyloplasts development. Therefore, the obstacle is worthy of further study.
The formation of pores in the endosperm of rice grains was related to the content of amylose/amylopectin, the location of a grain on the panicle and environmental factors.
4.4 Relationship between starch crystallinity and amylose content
In recent years, the XRD technology has been applied in starch research at home and abroad, especially in the studies on the industrial processing properties of starch 26-34. The contents of amylose and amylopectin were the most important factors determining the cooking and eating quality. This study found that the crystallinity of starch was negatively correlated with the content of amylose. In addition, the crystallinity of starch was correlated with the flowering sequence to a certain degree. However, glutinous rice contained little or none amylose and its starch crystallinity was not much higher than that of conventional indica and japonica rice. The results indicated that the relationship between starch crystallinity and amylopectin content was not a simple one-to-one corresponding relationship. The relationship between starch crystallinity and cooking and eating quality, the optimal starch crystallinity range, and the corresponding relationship between starch crystallinity of cultivars with similar amylopectin contents maybe hot spots in the future research on rice quality.