Recent Research Advances in the Development of Chalkiness and Transparency in Rice

Abstract

The appearance quality of rice represent the primary concern of consumers when choosing rice, as well as a necessary condition for high-quality rice. In the past, the focus of attention on rice appearance quality was mainly on chalkiness, and most previous reviews on rice appearance quality focused on the chalky phenotype of rice, while some more generalized chalkiness as the only indicator of rice appearance quality. This paper objectively analyses the definitions and interrelationships of rice appearance quality indicators at the present stage. Then, the formation mechanism and research status of rice appearance quality were analyzed from three aspects: endosperm structure, genetic background, and endosperm material basis. The two indicators (chalkiness and transparency) were selected, having the greatest influence on appearance, as the starting point. On this basis, the problems in the current research on rice appearance quality were analyzed and relevant suggestions are put forward, aiming to provide a theoretical basis for the overall improvement of rice appearance quality under large-scale production conditions.

1. Introduction

In China, rice is an indispensable staple food in people’s daily life. As people’s living standards improve, they pay more attention to the quality of the rice they buy, and thus high-quality rice means a higher commodity value. Obtaining rice with high yield and high quality is an important goal of rice breeding at present. High-quality rice involves many aspects, such as good appearance and good taste. Particularly, rice appearance is an important factor in determining the commercial value of rice and is also a trait of primary concern for consumers [1]. Therefore, it is significant to understand the development mechanism, genetic background, and influencing factors of rice appearance quality in order to improve the appearance quality of rice.

2. Definition and Interrelationship of Rice Appearance Quality Indicators

Rice appearance quality includes grain shape, chalkiness, and transparency. Grain shape is mainly used to indicate the shape and size of the grain, which can be measured by grain length, width and aspect ratio. Chalky areas refer to the white opaque areas in the rice grain, which can be phenotypically divided into white core, white belly, and white back. Chalkiness is usually used to measure chalky rice, which refers to the ratio of white opaque areas to the total area of the rice [2]. Rice transparency refers to the degree of transparency of fine rice, which is commonly measured by its transmittance. For the transparency trait of fine rice, some researchers classified transparency indicators into seven classes: fully transparent glassy, transparent, translucent, dark, off-white, light creamy white, and pure creamy white [3].

In general, long-thin rice has lower chalkiness than short round rice. This is mainly related to the grain filling patterns. During grain filling, nutrients can more easily reach the vicinity of the dorsal vascular bundle and tend to be less enriched away from the vascular bundle. Thus, short round rice is more likely to generate white belly and white core [4,5,6]. Rice transparency is affected by chalkiness and rice with higher chalkiness is generally less transparent. Rice with a better chalky phenotype only is likely to have a higher transparency phenotype, because transparency is also affected by multiple factors such as moisture content, fine rice texture and growth environment [7].

Consumers in different regions have different preferences for rice grain types due to cultural differences, but in terms of chalkiness and transparency, they all prefer rice with low chalkiness and high transparency [8,9]. Chalkiness is an important factor affecting the appearance and grading of rice and the impact of rice transparency (as an important component of rice appearance quality) on the commercial value of rice is increasingly emphasized. For example, a series of soft japonica rice varieties have been developed in the rice paddy field of the Yangtze River Delta region in recent years, and many japonica varieties have been recognized by the market for their excellent taste. However, compared with some traditional high-quality japonica rice, this soft japonica rice has unstable transparency traits, which restrict their market competitiveness. Therefore, a systematic study on the development and regulation mechanisms of rice chalkiness and transparency is important to improve the rice appearance quality. This can provide ideas for supporting cultivation practices applicable to improving the appearance of rice. The study can also provide a theoretical basis for breeders to select rice with better appearance. The aim is to develop domestic and international markets for high-quality rice [10]

3. Endosperm Structure for the Development of Rice Appearance Quality

On a microscale, rice appearance quality is the specific appearance characteristics developed after the involvement of rice grain endosperm in the light reflection and transmission, and it is mainly the fine endosperm structure that affects the light. Thus, it is important to explore the development of rice appearance quality based on the fundamentals of the rice grain structure. Currently, it has been found that starch, as the most important storage material in rice endosperm, accounts for over 80% of the dry weight of endosperm. Therefore, starch grain morphology and fine structure in the endosperm are important factors affecting the appearance quality of rice [11].

Many studies have demonstrated that the chalky phenotype is mainly an optical property due to the loose arrangement of regional starch grains in the endosperm [2,5,12]. Insufficient filling of the amyloplast results in the round and loosely distributed starch grains with air between them, which prevents light transmission by scattering and thus causes a chalky appearance. Insufficient amyloplast filling occurs in the belly, back, and core of rice, thus leading to the development of white belly, white back, and white core [13]. Many scholars have concluded that the development of rice chalkiness is closely related to the dynamic variation of dry matter accumulation, filling rate and water content of rice grains during rice filling [14,15].

Zhang et al. analyzed the structural relationship between transparency and endosperm starch grains of transgenic rice with different amylose content (AC) derived from glutinous rice. It is found that there were many cavities in the middle of the endosperm starch grains of glutinous rice and that the number of cavities showed a decreasing trend with the increase of AC. The transparency of rice was also improved. Thus, it is concluded that the opacity of glutinous rice and the translucency of soft japonica rice were both caused by cavities in the middle of the starch grain since the cavities caused light scattering and affected rice transparency [16,17]. Yang et al. stated that the formation of cavities in starch grains may be due to the gradual loss of water during the transition to the mature stage after starch grains reached a fixed number and shape and filled endosperm cells in the milk stage [18]. To further investigate the cavity formation of starch grains, Hao et al. conducted a structural analysis of the dynamic cross-section of starch grain development [11]. It is found that the cavity area in the single starch grain in the endosperm was formed about four days after flowering (DAF), and it showed a stable increase in size from 4 DAF to 20 DAF. At 25–30 DAF, the cavity area of single starch grains tended to stabilize.

4. Genetic Background of Rice Appearance Quality Formation

The chalk traits in rice have a complicated genetic mechanism. They are mainly controlled by quantitative traits and interact with environmental factors. It is difficult to analyze the genetic mechanism of chalky traits by traditional analysis methods. Thus, quantitative trait loci (QTL) analysis of chalky traits in rice is commonly performed and has also made great progress. The genetic regulation and formation mechanism of rice transparency has been rarely studied and no clear direct regulatory genes have been identified. Many genes have been identified that have indirect regulatory effects on rice appearance, such as starch synthesis-related genes. These genes related to amylose and amylopectin synthesis are not directly related to rice appearance, but their functional loss, repression, or changes in expression can ultimately affect the appearance quality of rice. Starch synthesis is controlled by different subtypes of multiple enzymes, and the action of one or more enzymes in starch synthesis will induce different morphological starch structures, resulting in different rice appearance phenotypes. In addition, mutations in transcription factors that regulate starch synthesis genes can also cause opaque rice grains [19].

4.1. Genetic Analysis of Appearance Quality

Chalky traits of rice are mainly classified into chalkiness, chalky area, chalky rate, white core area, white core rate, white back area and white rate back, etc. Currently, QTLs associated with chalky traits have been identified on all 12 chromosomes of rice. Chalk5 is the first cloned major QTL for chalkiness, which affects the regular accumulation of starch grains and proteasomes by influencing the pH balance of the endosomal transport system during development [20]. Recently Zhu et al., localized a QTL for chalkiness on chromosome 1 called qPCG1 with a physical distance of about 139 kb, explaining 6.8–21.9% of the phenotypic variation in the heterogeneous region of chromosome 1 [21]. Yang et al. analyzed two closely linked QTLs controlling chalkiness and grain shape on chromosome 8. qPGC8.1 was located in the 1382.6 kb interval and qPGC8.2 in the 2057.1 kb interval. qPGC8.1 controlled seed chalkiness and grain width, while qPGC8.2 was responsible for seed chalkiness, grain length and grain width [22]. Yang et al. also localized two QTLs associated with chalkiness on chromosomes 9 and 11 [23]. Tan et al. detected two white-belly QTLs located in the interval RG360-C734a on chromosome 5 and the interval R1245-R1789 on chromosome 7 in a recombinant inbred line derived from Zhenshan 97/Minghui 63, and the effect of the former QTL accounted for 87.2% of the variation, respectively. Two core-white QTLs were detected in the interval RG6360-C734a on chromosome 5 and in the interval Wx-R1952 on chromosome 6, which accounted for 11.6% and 7.5% of the variation, respectively [24]. He et al., detected QTLs controlling chalky traits on chromosomes 3, 8, and 12 using the doubled haploid (DH) population, respectively [25]. These results indicate that QTLs for chalkiness are mainly distributed on chromosomes 5, 6, 7, and 8 of rice, and the above QTLs are stable under different genetic backgrounds and environments. The contribution of the chalkiness QTLs to the phenotype is influenced by genetic background and environmental conditions.

4.2. Appearance Quality-Associated Starch-Regulated Genes

There are five major types of key enzymes for starch synthesis: adenosine diphosphate glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), soluble starch synthase (SSS), starch branching enzyme (SBE), and starch debranching enzyme (DBE) [26].

In the first step in catalyzing starch biosynthesis, glucose-1-phosphate (G1P) and ATP are catalyzed by AGPase to produce the activated glucosyl donor ADP-glucose (ADP-Glc) and inorganic pyrophosphate (PPi).

The waxy gene (Wx) of rice encodes the granule-bound starch synthase I (GBSSI) that is required for the synthesis of amylose in the endosperm and is the major gene controlling amylose synthesis with the highest expression at mid-endosperm development [27].

There are four SSS (i.e., SSⅠ, SSⅡ, SSⅢ and SSⅣ) in rice, including eight isozymes (i.e., SSⅠ, SSⅡa, SSⅡb, SSⅡc, SSⅢa, SSⅢb, SSⅣa and SSⅣb), which are highly expressed in the early stages of endosperm development [27]. Previous studies on the genetics and biochemistry of a large number of cereal mutants revealed that SSI is responsible for the extension of short chains on amylopectin, SSII for the extension of medium and long chains, SSIII for the extension of long chains, and SSIV may be involved in short chains of glucose and starch granule initiation. The differential expression of SSS has an important effect on the amylopectin structure.

In rice, SBE contains both SBEⅠ and SBEⅡ. SBEⅡ is divided into two isoforms in rice grains, i.e., SBEⅡa and SBEⅡb. SBEⅡa is widely present in every tissue, while SBEⅡb is specifically expressed only in the endosperm [25]. SBEⅡb acts only on the short chain of DP6~7, and SBEⅡa and SBEⅡb can break down the outer and inner chains of branched glucan, respectively, to form the short and medium chains of DP ≤ 40 [28]. General inhibition or mutation of SBE genes is an effective method for developing high-amylose cereal crops [29]. Starch branching enzymes are the only enzymes for the formation of branched chains of amylopectin.

The role of DBE is to hydrolyze the α-1,6 glycosidic bond and may act as a modifier of the branching structure of starch. There are two types of debranching enzymes in plants: isoamylases (ISA, ISA-1, ISA-2, ISA-3) that can modify amylopectin and phytoglycogen, and pullulanases (acting only on amylopectin and pullulan) [30,31]. Amylopectin is synthesized by the combined catalysis of AGPase, SSS, SBE, and DBE. SBEs alone cannot catalyze the formation of regular clusters of amylopectin, and the randomly formed branched chains block the further contact of SBEs and SSS to the substrate. DBEs can selectively remove improperly positioned branches, resulting in the formation of clusters of branched chains [27]. Many researchers have proposed that amylose biosynthesis is catalyzed by DBE and SSS using branches of amylopectin as substrates (primers) [32].

Fujita et al. studied the mutants ss3a-1 and ss3a-2 with SSIIIa loss resulting in chalkiness and found round amyloplasts with loose packing and significant chalkiness in both mutants, indicating that the functional loss of SSS directly leads to increased chalkiness [33]. Zhang et al. used RNA interference to obtain SSIIa/SSIIIa double mutant plants, which had a distinct chalky phenotype with increased AC, decreased short (degree of polymerization (DP5–6) and long (DP12–23) amylose, decreased amylopectin, and increased amylose with medium-long DP. Comparative analysis between single and double mutant plants revealed that SSIIa and SSIIIa interacted in starch synthesis, altering the fine structure of amylopectin and thus resulting in the chalky trait [34]. Fujita crossed the ss1 deletion with the ss3a deletion mutant to obtain the genotypes ssIssI/SS3ass3a and SSIssI/ss3ass3a F2 plants, with distinctly chalky progeny. The progeny had high straight-chain starch content in the endosperm, rounded starch granules and significantly higher expression of starch granule synthase (GBSSI) and ADP-glucose phosphorylase (AGPase) than the wild type and parental mutant material [35]. The above study shows that SSI, SSII, and SSIIIS genes play an important role in starch synthesis by altering the amount or structure of amylopectin and thus changing the physical traits of starch granules, which in turn regulates the development of chalky traits. Tanaka et al. found that the BEIIb deletion mutant, EM10, had smaller grains and a significantly chalky endosperm.

In previous studies on some dark endosperm mutant rice, it is found that some low enzymatic activity natural mutations or genetically modified low enzymatic activity proteins of GBSSI caused the rice to exhibit a translucent phenotype due to the synthesis of less amylose (below 13%). This suggests a direct correlation between GBSSI and rice transparency [36,37,38]. Zhang et al. studied transgenic rice lines with GBSSI activity and found that AC was lower in rice lines with lower GBSSI activity, and the grains showed a milky opaque appearance. It is also found that GBSSI was not only responsible for the synthesis of amylose starch but also involved in the biosynthesis of medium and extra-long branched chains. This in turn affected rice grain transparency, thermodynamic and gelatinization characteristics, and crystal structure [17]. Liu et al., analyzed OsGBSS1 activity and AC in homozygous transgenic lines with mutations in the OsGBSS1 (Wx) gene and found that it is feasible to finely regulate the AC of rice grains by modifying OsGBSS1 activity [39]. This may provide a reference for achieving synergistic enhancement of rice appearance and palatability to some extent through precise regulation of AC. Li et al. obtained new low-AC japonica rice with low AC and transparent appearance by knocking down SSSII-2, a gene encoding the SSS subtype in Nipponbare (Nip). The rice had excellent palatability and remained transparent in appearance under low moisture conditions, with fewer cavities in the starch grains and higher starch crystallinity than the control. This study indicates that the introduction of the SSII-2 RNAi reduced the AC of rice while substantially improving rice transparency, suggesting that the SSSII-2 gene may be an important gene that controls starch grain morphology and fine structure and thus affects rice transparency traits [40]. Then, Hao et al. further investigated the effect of SSII-2 RNAi in rice with lower AC on rice transparency and found that SSII-2 RNAi improved rice transparency to a limited extent when AC was below 9%. Particularly, the knockdown and interference of the SSII-2 gene in parents under lower AC conditions did not improve transparency significantly, while the knockdown of the SSII-3 gene in parents was able to improve rice transparency to some extent [11]. This study suggests that the effect of expression of homozygous SSS genes on rice transparency may differ in different Wx allele backgrounds, and the effect of the SSII-3 gene on transparency was higher than that of the SSII-2 gene at low AC.

4.3. Other Genes Associated with Appearance Quality Formation

Sucrase is also important for starch formation during rice grain development. Sucrase is located in the cell wall and its function is to convert sucrose into starch-making substances; inactive sucrase causes insufficient rice grain filling. When studying the GIF1 gene mutant, it is found that the sucrase activity was deficient and was only 17% of that of normal plants. The accumulation of sucrose in the vascular and floral bundles was inadequate. The accumulation of starch in the endosperm during the filling period was inadequate. Moreover, the mutant had a lower content of both amylose and amylopectin than wild plants and a loose starch structure, thus producing more chalky grains [41]. This suggests that GIF1 regulates the development of chalkiness by regulating sucrase activity. Some genes associated with chalkiness were also identified in the mutants studied for the floury chalky phenotype. Flo2 was involved in regulating the synthesis of storage proteins and storage starch in rice endosperm. The Flo2 mutant grains had a floury chalky phenotype with loosely distributed starch grains and the RT-PCR analysis shows that the genes related to starch synthesis (e.g., BEI, BEIIb, and AGPases) were reduced. It is found that Flo2 expression was significantly reduced at a higher temperature, and therefore increasing Flo2 expression under high-temperature conditions could improve chalky traits [42]. In addition, Kang et al. used T-DNA insertion mutants to obtain the floury core white mutant, Flo4, which had loosely packed starch grains, decreased AC, decreased amylose, decreased amylose/amylopectin ratio, increased lipid content and significant chalkiness [41]. In addition to the sucrose-regulated pathway, Wu et al. cloned the F-box gene WCR1, which negatively regulates rice chalkiness. WCR1 positively affects the transcription of and interacts with the metallothionein MT2b. This inhibits 26S proteasome-mediated degradation and leads to a reduction in reactive oxygen species and programmed delayed death of rice endosperm cells. Then, the chalkiness is reduced [43]. OsbZIP60 is an important regulator of rice seed chalking. Genetic analysis showed that the knockdown of OsbZIP60 resulted in high seed chalking and abnormal storage material structure, and the expression of unfolded protein response (UPR) genes. For example, OsbZIP50, OsBiP1, OsBiP2, and OsBiP3 were up-regulated in endosperm cells with OsbZIP60, and all overexpression of these UPR genes resulted in varying degrees of chalkiness [44]. The mutant with deletion of the plastid phosphorylase gene (Pho1) was studied and found to have folded grains, reduced and irregularly rounded starch grains, an increased proportion of short amylopectin in the endosperm (DP ≤ 11), and chalky endosperm. Particularly, grain folds and chalkiness showed a significant increase at low temperatures [45].

Dull mutants refer to mutant types with a dark opaque endosperm phenotype. These mutants generally have lower AC than the current soft japonica rice and the dark endosperm phenotype is revealed without prolonged aging or reduced moisture treatment. Genetic analysis shows that the low AC of dull mutants is generally controlled by a cryptic single gene (Du) independent of Wx, and Dull mutants are mainly obtained by chemical and radiation mutagenesis. Twelve genes controlling the dull locus have been reported in rice. Dull genes can indirectly regulate the biosynthesis of amylose and thus alter the physicochemical properties of rice. Previous studies found that Du-1, Du-2, and Du-3 encode regulatory splicing factors similar to SR proteins, which affect the slicing efficiency of Wxb precursors and RNA translation and stability, resulting in the dull phenotype [46]. Wang et al. studied the novel dull mutant w54 and found that the Wx gene transcript level and protein content of w54 decreased. This may be attributed to the fact that the mutation of the w54 gene affected the transcription and splicing of Wx, which in turn regulated the synthesis of amylose and amylopectin, and ultimately affected starch biosynthesis [47]. Therefore, the dull gene affects rice transparency mainly by affecting the expression of the Wx gene, thereby regulating the AC and developing the dark endosperm appearance.

In addition to gene down-regulation and overexpression, better crop improvement may have to be achieved through post-translational site modifications or regulatory enzymes. Understanding and regulating the starch synthesis and thus determining rice appearance quality is a complex process, and more mutants are needed to reveal the regulatory mechanisms.

5. Endosperm Material for the Formation of Rice Appearance Quality

Based on the analysis of endosperm composition, previous studies found that different moisture contents, as well as amylose and protein in rice endosperm, can have a large impact on the appearance quality of rice.

5.1. Moisture

According to the classification based on AC, japonica rice with AC ranging from 8% to 16% is called soft japonica rice [48]. However, it has been found that some soft japonica rice seeds develop a dark endosperm phenotype, and the appearance quality is significantly reduced after storage at a specified moisture content for some time after harvest [49]. Li et al., found that soft japonica rice varieties (Nanjaponica 46 and Guangdong 194) showed a significant decrease in grain endosperm transparency at storage moisture below 12% [40]. Lu et al., found that the endosperm of soft japonica rice started to change from transparent to opaque after 6 h of drying at 40 °C by gradient drying experiments, while no significant changes occurred in common japonica rice [1]. Zhang et al., also found that controlling grain moisture can regulate the transparency of soft japonica rice grains, confirming the positive effect of moisture content on the transparency of soft japonica rice [49]. It has also been found that this cloudy rice appearance caused by reduced moisture content is more “masked” in expression, usually appearing after a period of drying [50]. Numerous studies have shown that the transparency of some soft japonica rice decreases sharply with decreasing moisture content, but the chalkiness phenotype of rice does not change with changes in grain moisture content [1,49]. Therefore, rice grain moisture content can greatly affect the appearance quality of rice by regulating transparency. Currently, soft japonica rice is usually sold in the market by vacuum packaging to maintain a certain amount of moisture in order to improve its appearance quality when circulating in the market.

It is also found that the transparency of rice and the effect of moisture content on transparency show a significant positive correlation with the size of starch grain cavities [11,49]. Regarding the action mechanism between moisture, cavity and transparency, the authors suggest that the internal cavity of the starch grain could be filled by moisture at higher moisture content and thus does not affect the overall transparency of the endosperm. When the moisture content decreases, the water in the cavities is more easily precipitated than the semi-free water closely bound to the starch, and the cavities refract light to reduce the endosperm transparency. Larger cavities in the starch grain cause larger effects of moisture content on the transparency, which is also consistent with previous studies. The presence of cavities not only affects the appearance of rice, but also reduces the amount of starch that could be stored in the endosperm, which to some extent affects the yield and quality of rice.

5.2. Amylose

In fine rice with a moisture content of 14.0%, starch accounts for 76.7–78.4% of the total endosperm mass. Therefore, starch is the most important component of rice, and many previous studies have shown that the AC is an important factor affecting rice transparency. Zhang et al., investigated the transparency of dried brown rice with the same genotype but different AC and found that the transparency of rice grains at the same moisture content was significantly and positively correlated with AC [49]. Lu et al. analyzed the appearance of japonica rice with three types of AC (i.e., medium, low and glutinous) at a moisture content of 12% and found that the difference in the transparency of different rice varieties may be directly related to AC. Wu et al., found that the endosperm of all low AC materials showed easily identifiable cloudy traits under three months of indoor storage. However, the endosperm of medium or high AC varieties remained transparent in appearance. This material included early indica varieties (lines), inbred rice sterile lines and restorers [50]. This suggests that low AC may not only be able to affect the appearance quality of japonica rice, but all types of rice generally show reduced endosperm transparency at reduced AC. As mentioned above, more studies show a direct relationship between AC content and the number of pores in the starch grains [37,51,52,53]. Therefore, it is possible that rice AC content first affects the size and number of starch grain pores, and then the pores affect rice grain transparency under the synergistic effect of water. Currently, although increasing rice AC may be an effective measure to improve rice transparency, it may affect rice palatability. Therefore, further research is needed to synergistically improve the appearance and taste quality of rice.

5.3. Protein

Protein content in rice is second only to starch, and protein is not only an important nutritional quality indicator of rice but also has a large influence on the appearance quality of rice. Shi et al. investigated the effect of protein in rice quality determination through near-isogenic lines and different rice varieties. It is found that protein content was negatively correlated with chalky grain rate and chalkiness in both indica and japonica rice, indicating that an increase in protein content tended to improve the appearance quality [54]. Li et al., analyzed the relevant quality traits of japonica rice with high and low chalkiness, respectively, and concluded that chalkiness was negatively correlated with brown rice protein content in high chalkiness rice varieties [55]. Chun et al., found that the protein content of high chalkiness rice was higher than that of low chalkiness rice [56]. By investigating the relationship between rice appearance quality and protein fraction, Tong et al., found that gluten content showed a significant negative correlation with chalky grain rate and chalkiness, while albumin and globulin content had slight effects on appearance quality [57]. Wang et al., analyzed 8390 rice materials collected from all over China and found that protein content was positively correlated with transparency in indica rice and chalky grain rate and chalkiness in conventional indica rice while showing a negative correlation with chalky grain rate and chalkiness in hybrid indica rice. In japonica rice, the protein content was positively correlated with chalkiness and transparency in both conventional and hybrid japonica rice [58]. It has been shown that the protein content of rice shows a highly significant positive correlation with rice transparency. High protein content has improved the appearance quality of rice, probably because the protein body particles contained in rice fill the starch grains in the endosperm. Then, the refraction of light through the gaps of rice grains due to the loose filling of starch grains was reduced or eliminated. Thus, the appearance quality of rice was improved [59].

For rice, it has been shown that the level of protein content can affect the color and luster of rice. High-protein rice is generally more transparent than low-protein rice, with a slightly creamy tint. For cold rice, there is a certain negative correlation between protein and luster [60].

6. Conclusions and Outlook

The improvement of rice appearance quality must be a process that considers both chalky phenotypes and transparent phenotypes. Therefore, all the indicators of appearance quality should be considered comprehensively to improve the appearance of rice. In this way, the rice can better meet the standards of high-quality rice and adapt to the consumer market.

Many studies have been conducted at home and abroad on the development and regulation of the appearance quality of rice, mostly on the effects of endosperm composition and starch fine structure on the appearance quality of rice. Some correlations between endosperm components, appearance quality, and the development mechanism of appearance quality at the level of the starch structure have been obtained. In recent years, with the development and improvement of genetic engineering, there has also been research progress made regarding the relationship between gene expression related to starch synthesis and the appearance quality of rice. In addition, the appearance quality of rice is also highly susceptible to the influence of environmental factors. The type and expression of genes regulating rice appearance traits may also vary under different environmental conditions. Therefore, rational cultivation measures can be taken based on the starch fine structure and endosperm substance regulation. These measures can help to ensure the temperature, light, and nutrients required for the formation of high-quality starch at the stage of filling and maturity. Thus, this will provide the basis for the rational formation and arrangement of endosperm starch, and finally help to achieve the purpose of both increasing rice yield and improving rice appearance quality. Furthermore, breeders need to further explore genes related to appearance quality and their mechanisms of action. Based on the understanding of the genetic background of the breeding material for appearance quality, we can select high quality rice with a good genetic base, according to the cultivation environment.

Author Contributions

Writing—original draft preparation, P.F.; consulting references, J.X., G.L., J.T. and Z.Z.; funding acquisition, H.W. and H.Z. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We are grateful for grants from the National Natural Science Foundation of China, the National Modern Agricultural Industry Technology System Construction Special Project and Technology, the Jiangsu Modern Agricultural Industry Technology System Construction Project, the Jiangsu Provincial Agricultural Science and Technology Independent Innovation Fund Project, the Jiangsu Province Postgraduate Research Innovation Fund. We would like to thank the editor and the reviewers for their useful feedback that improved this paper.

Conflicts of Interest

The authors declare no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

1. Lu, Y.; Zhang, X.M.; Qi, Y.; Zhang, C.Q.; Ling, Y.P.; Liu, Q.Q. Scanning electron microscopy (SEM) analysis of rice grains with different transparency. Chin. Rice Sci.; 2018; 32, pp. 189-199.

2. Liao, B.; Zhang, G. Advances in chalkiness of rice. Crop Res.; 2015; 29, pp. 77-83.

3. Shi, C.H.; Wu, J.G.; Fan, L.J.; Zhu, J.; Wu, P. Developmental genetic analysis of rice transparency under different environmental conditions. Genetics; 2002; 29, pp. 56-61.

4. Guo, Z.K. Genotypic Differences and Nitrogen Effects on Appearance Quality and Milling Quality of Japonica Rice Varieties Mainly Cultivated in Jiangsu Province. Master’s Thesis; Nanjing Agricultural University: Nanjing, China, 2015.

5. Chen, F.; Wang, H.F.; Gao, J.; Zhou, X.B.; Xin, C.Y.; Zhang, Y.S.; Xu, J.D.; Yuan, S.J. Advances in chalkiness traits of rice. Barley Cereal Sci.; 2018; 35, pp. 7-10.

6. Peng, B.; Guang, H. Research progress of chalkiness formation in rice. Crop Res.; 2009; 23, pp. 310-313.

7. Nevame, A.Y.M.; Emon, R.M.; Malek, M.A.; Hasan, M.M.; Alam, M.A.; Muharam, F.M.; Aslani, F.; Rafii, M.Y.; Ismail, M.R. Relationship between High Temperature and Formation of Chalkiness and Their Effects on Quality of Rice. Biomed Res. Int.; 2018; 2018, 1653721. [DOI: https://dx.doi.org/10.1155/2018/1653721]

8. Sreenivasulu, N.; Butardo, V.M.; Misra, G.; Cuevas, R.P.; Anacleto, R.; Kishor, P.B.K. Designing climate-resilient rice with ideal grain quality suited for high-temperature stress. J. Exp. Bot.; 2015; 66, pp. 1737-1748. [DOI: https://dx.doi.org/10.1093/jxb/eru544]

9. Wang, Y.; Wang, Y.; Sun, X.; Caiji, Z.; Yang, J.; Cui, D.; Cao, G.; Ma, X.; Han, B.; Xue, D. et al. Influence of ethnic traditional cultures on genetic diversity of rice landraces under on-farm conservation in southwest China. J. Ethnobiol. Ethnomed.; 2016; 12, 51. [DOI: https://dx.doi.org/10.1186/s13002-016-0120-0]

10. Zhu, D.W.; Zhang, H.C.; Guo, B.W.; Dai, Q.G.; Huo, Z.Y.; Xu, K.; Wei, H.Y.; Gao, H. Development and prospect of Chinese soft rice. J. Yangzhou Univ.; 2015; 36, pp. 47-52.

11. Hao, W.Z. Study on the Structural basis and Genetic Regulation of Starch Transparency Formation in Rice. Master’s Thesis; Yangzhou University: Yangzhou, China, 2021.

12. Zheng, Y.; Zeng, D.; Wei, H.; Yuan, X.U.; Yunjie, G.U.; Wang, Z. Structure Observation of Rice Endosperm Tissues. Chin. J. Rice Sci.; 2017; 31, pp. 91-98. (In Chinese)

13. Liu, Y.C.; Shao, G.N.; Hu, P.S. Advances in chalkiness traits of rice. Chin. Rice; 2012; 18, pp. 1-8.

14. Liu, X.; Fu, Y.P.; Zhu, Y.R.; Li, Y.P.; Wang, Y. Physiological and genetic mechanisms of chalkiness formation in rice. Plant Physiol. Commun.; 2007; 43, pp. 569-574.

15. Yang, F.; Song, H.; Cui, X.Y.; Ma, J.Y. Comparative study on grain-filling dynamics of different chalky japonica rice. J. Jilin Agric. Univ.; 2003; 25, pp. 13-17.

16. Zhang, C.; Zhu, L.; Shao, K.; Gu, M.; Liu, Q. Toward underlying reasons for rice starches having low viscosity and high amylose: Physiochemical and structural characteristics. J. Sci. Food Agric.; 2013; 93, pp. 1543-1551. [DOI: https://dx.doi.org/10.1002/jsfa.5987]

17. Zhang, C.; Chen, S.; Ren, X.; Lu, Y.; Liu, D.; Ca, X.; Li, Q.; Gao, J.; Liu, Q. Molecular Structure and Physicochemical Properties of Starches from Rice with Different Amylose Contents Resulting from Modification of OsGBSSI Activity. J. Agric. Food Chem.; 2017; 65, pp. 2222-2232. [DOI: https://dx.doi.org/10.1021/acs.jafc.6b05448]

18. Yang, X.Y. Analysis of Physicochemical Basis of Quality Formation of Japonica Rice Based on Starch Fine Structure. Ph.D. Thesis; Nanjing Agricultural University: Nanjing, China, 2015.

19. Wang, J.; Xu, H.; Zhu, Y.; Liu, Q.; Cai, X. OsbZIP58, a basic leucine zipper transcription factor, regulates starch biosynthesis in rice endosperm. J. Exp. Bot.; 2013; 64, pp. 3453-3466. [DOI: https://dx.doi.org/10.1093/jxb/ert187]

20. Li, Y.; Fan, C.; Xing, Y.; Yun, P.; Luo, L.; Yan, B.; Peng, B.; Xie, W.; Wang, G.; Li, X. et al. Chalk5 encodes a vacuolar H+-translocating pyrophosphatase influencing grain chalkiness in rice. Nat. Genet.; 2014; 46, 398.Erratum in Nat. Genet. 2014, 46, 657 [DOI: https://dx.doi.org/10.1038/ng.2923]

21. Zhu, A.; Zhang, Y.; Zhang, Z.; Wang, B.; Xue, P.; Cao, Y.; Chen, Y.; Li, Z.; Liu, Q.; Cheng, S. et al. Genetic Dissection of qPCG1 for a Quantitative Trait Locus for Percentage of Chalky Grain in Rice (Oryza sativa L.). Front. Plant Sci.; 2018; 9, 1173. [DOI: https://dx.doi.org/10.3389/fpls.2018.01173]

22. Yang, W.F.; Xiong, L.; Liang, J.Y.; Hao, Q.W.; Luan, X.; Tan, Q.Y.; Lin, S.W.; Zhu, H.T.; Liu, G.F.; Liu, Z.P. et al. Substitution Mapping of Two Closely Linked QTLs on Chromosome 8 Controlling Grain Chalkiness in Rice. Rice; 2021; 14, 85. [DOI: https://dx.doi.org/10.1186/s12284-021-00526-4]

23. Yang, W.F.; Liang, J.Y.; Hao, Q.W.; Luan, X.; Tan, Q.Y.; Lin, S.W.; Zhu, H.T.; Liu, G.F.; Liu, Z.P.; Bu, S.H. et al. Fine mapping of two grain chalkiness QTLs sensitive to high temperature in rice. Rice; 2021; 14, 33. [DOI: https://dx.doi.org/10.1186/s12284-021-00476-x]

24. Tan, Y.F.; Xing, Y.Z.; Li, J.X.; Yu, S.B.; Xu, C.G.; Zhang, Q.F. Genetic bases of appearance quality of rice grains in Shanyou 63, an elite rice hybrid. Theor. Appl. Genet.; 2000; 101, pp. 823-829. [DOI: https://dx.doi.org/10.1007/s001220051549]

25. He, P.; Li, S.G.; Qian, Q.; Ma, Y.Q.; Li, J.Z.; Wang, W.M.; Chen, Y.; Zhu, L.H. Genetic analysis of rice grain quality. Theor. Appl. Genet.; 1999; 98, pp. 502-508. [DOI: https://dx.doi.org/10.1007/s001220051098]

26. Huaxin, H.; Chuantian, Y.; Jihui, Z.; Lixia, Z.; Yeming, B.; Enpeng, L.; Robert, G.G. Competition between Granule Bound Starch Synthase and Starch Branching Enzyme in Starch Biosynthesis. Rice; 2019; 12, 96.

27. Lv, B.; Guo, Z.G.; Liang, J.S. Effects of the activities of key enzymes involved in starch biosynthesis on the fine structure of amylopectin in developing rice (Oryza sativa L.) endosperms. Sci. China Ser. C Life Sci.; 2008; 51, pp. 863-871.

28. Nakamura, Y.; Utsumi, Y.; Sawada, T.; Aihara, S.; Utsumi, C.; Yoshida, M.; Kitamura, S. Characterization of the Reactions of Starch Branching Enzymes from Rice Endosperm. Plant Cell Physiol.; 2010; 51, pp. 776-794. [DOI: https://dx.doi.org/10.1093/pcp/pcq035]

29. Wang, J.; Hu, P.; Chen, Z.; Liu, Q.; Wei, C. Progress in High-Amylose Cereal Crops through Inactivation of Starch Branching Enzymes. Front. Plant Sci.; 2017; 8, 469. [DOI: https://dx.doi.org/10.3389/fpls.2017.00469]

30. Pandey, M.K.; Rani, N.S.; Madhav, M.S.; Sundaram, R.M.; Varaprasad, G.S.; Sivaranjani, A.K.P.; Bohra, A.; Kumar, G.R.; Kumar, A. Different isoforms of starch-synthesizing enzymes controlling amylose and amylopectin content in rice (Oryza sativa L.). Biotechnol. Adv.; 2012; 30, pp. 1697-1706. [DOI: https://dx.doi.org/10.1016/j.biotechadv.2012.08.011]

31. Lin, L.; Huang, J.; Zhang, L.; Liu, Q.; Wei, C. Effects of inhibition of starch branching enzymes on starch ordered structure and component accumulation in developing kernels of rice. J. Cereal Sci.; 2020; 91, 102884. [DOI: https://dx.doi.org/10.1016/j.jcs.2019.102884]

32. Van De Wal, M.; D’Hulst, C.; Vincken, J.; Buleon, A.; Visser, R.; Ball, S. Amylose is synthesized in vitro by extension of and cleavage from amylopectin. J. Biol. Chem.; 1998; 273, pp. 22232-22240. [DOI: https://dx.doi.org/10.1074/jbc.273.35.22232]

33. Fujita, N.; Yoshida, M.; Kondo, T.; Saito, K.; Utsumi, Y.; Tokunaga, T.; Nishi, A.; Satoh, H.; Park, J.; Jane, J. et al. Characterization of SSIIIa-Deficient mutants of rice: The function of SSIIIa and pleiotropic effects by SSIIIa deficiency in the rice endosperm. Plant Physiol.; 2007; 144, pp. 2009-2023. [DOI: https://dx.doi.org/10.1104/pp.107.102533]

34. Zhang, G.; Cheng, Z.; Zhang, X.; Guo, X.; Su, N.; Jiang, L.; Mao, L.; Wan, J. Double repression of soluble starch synthase genes SSIIa and SSIIIa in rice (Oryza sativa L.) uncovers interactive effects on the physicochemical properties of starch. Genome; 2011; 54, pp. 448-459. [DOI: https://dx.doi.org/10.1139/g11-010]

35. Fujita, N.; Satoh, R.; Hayashi, A.; Kodama, M.; Itoh, R.; Aihara, S.; Nakamura, Y. Starch biosynthesis in rice endosperm requires the presence of either starch synthase I or IIIa. J. Exp. Bot.; 2011; 62, pp. 4819-4831. [DOI: https://dx.doi.org/10.1093/jxb/err125]

36. Mikami, I.; Uwatoko, N.; Ikeda, Y.; Yamaguchi, J.; Hirano, H.Y.; Suzuki, Y.; Sano, Y. Allelic diversification at the wx locus in landraces of Asian rice. Theor. Appl. Genet.; 2008; 116, pp. 979-989. [DOI: https://dx.doi.org/10.1007/s00122-008-0729-z]

37. Adegoke, T.V.; Wang, Y.; Chen, L.; Wang, H.; Liu, W.; Liu, X.; Cheng, Y.; Tong, X.; Ying, J.; Zhang, J. Posttranslational Modification of Waxy to Genetically Improve Starch Quality in Rice Grain. Int. J. Mol. Sci.; 2021; 22, 4845. [DOI: https://dx.doi.org/10.3390/ijms22094845]

38. Syahariza, Z.A.; Sar, S.; Warren, F.J.; Zou, W.; Hasjim, J.; Tizzotti, M.J.; Gilbert, R.G. The importance of amylose and amylopectin fine structures for starch digestibility in cooked rice grains. Food Chem.; 2013; 136, 742.Erratum in Food Chem. 2014, 145, 617–618 [DOI: https://dx.doi.org/10.1016/j.foodchem.2012.08.053]

39. Liu, D.; Wang, W.; Cai, X. Modulation of amylose content by structure-based modification of OsGBSS1 activity in rice (Oryza sativa L.). Plant Biotechnol. J.; 2014; 12, pp. 1297-1307. [DOI: https://dx.doi.org/10.1111/pbi.12228]

40. Li, Q.; Huang, L.; Chu, R.; Li, J.; Jiang, M.; Zhang, C.; Fan, X.; Yu, H.; Gu, M.; Liu, Q. Down-Regulation of SSSII-2 Gene Expression Results in Novel Low-Amylose Rice with Soft, Transparent Grains. J. Agric. Food. Chem.; 2018; 66, pp. 9750-9760. [DOI: https://dx.doi.org/10.1021/acs.jafc.8b02913]

41. Kang, H.G.; Park, S.; Matsuoka, M.; An, G.H. White-core endosperm floury endosperm-4 in rice is generated by knockout mutations in the C-4-type pyruvate orthophosphate dikinase gene (OsPPDKB). Plant J.; 2005; 42, pp. 901-911. [DOI: https://dx.doi.org/10.1111/j.1365-313X.2005.02423.x]

42. She, K.-C.; Kusano, H.; Koizumi, K.; Yamakawa, H.; Hakata, M.; Imamura, T.; Fukuda, M.; Naito, N.; Tsurumaki, Y.; Yaeshima, M. et al. A Novel Factor FLOURY ENDOSPERM2 Is Involved in Regulation of Rice Grain Size and Starch Quality. Plant Cell; 2010; 22, pp. 3280-3294. [DOI: https://dx.doi.org/10.1105/tpc.109.070821]

43. Wu, B.; Yun, P.; Zhou, H.; Xia, D.; Gu, Y.; Li, P.B.; Yao, J.L.; Zhou, Z.Q.; Chen, J.X.; Liu, R.J. et al. Natural variation in WHITE-CORE RATE 1 regulates redox homeostasis in rice endosperm to affect grain quality. Plant Cell; 2022; 34, pp. 1912-1932. [DOI: https://dx.doi.org/10.1093/plcell/koac057]

44. Yang, W.; Xu, P.; Zhang, J.; Zhang, S.; Li, Z.; Yang, K.; Chang, X.; Li, Y. OsbZIP60-mediated unfolded protein response regulates grain chalkiness in rice. J. Genet. Genom.; 2022; 49, pp. 414-426. [DOI: https://dx.doi.org/10.1016/j.jgg.2022.02.002]

45. Satoh, H.; Shibahara, K.; Tokunaga, T.; Nishi, A.; Tasaki, M.; Hwang, S.; Okita, T.W.; Kaneko, N.; Fujita, N.; Yoshida, M. et al. Mutation of the plastidial α-Glucan phosphorylase gene in rice affects the synthesis and structure of starch in the endosperm. Plant Cell; 2008; 20, pp. 1833-1849. [DOI: https://dx.doi.org/10.1105/tpc.107.054007]

46. Isshiki, M.; Matsuda, Y.; Takasaki, A.; Wong, H.L.; Satoh, H.; Shimamoto, K. Du3, a mRNA cap-binding protein gene, regulates amylose content in Japonica rice seeds. Plant Biotechnol.-Nar.; 2008; 25, pp. 483-487. [DOI: https://dx.doi.org/10.5511/plantbiotechnology.25.483]

47. Wang, P. Phenotypic Analysis and Gene Mapping of Rice Dark Endosperm Mutant W. Master’s Thesis; Nanjing Agricultural University: Nanjing, China, 2015.

48. Xu, Z.Z.; Mao, X.Q.; Chen, S.W.; Yang, L. Quality analysis and identification of yunnan soft rice cultivars. J. Shandong Agric. Univ.; 2002; 3, pp. 380-383.

49. Zhang, L.; Zhao, L.; Zhang, J.; Cai, X.; Liu, Q.; Wei, C. Relationships between transparency, amylose content, starch cavity, and moisture of brown rice kernels. J. Cereal Sci.; 2019; 90, 102854. [DOI: https://dx.doi.org/10.1016/j.jcs.2019.102854]

50. Wu, D.X.; Xia, Y.W.; Li, X.C. Analysis on forming basis and rapid recognition condition of appearance cloud and fog in rice endosperm. Chin. Rice Sci.; 2001; 3, pp. 33-37.

51. Bao, J.; Ying, Y.; Zhou, X.; Xu, Y.; Wu, P.; Xu, F.; Pang, Y. Relationships among starch biosynthesizing protein content, fine structure and functionality in rice. Carbohyd. Polym.; 2020; 237, 116118. [DOI: https://dx.doi.org/10.1016/j.carbpol.2020.116118]

52. Lin, Z.; Zheng, D.; Zhang, X.; Wang, Z.; Lei, J.; Liu, Z.; Li, G.; Wang, S.; Ding, Y. Chalky part differs in chemical composition from translucent part of japonica rice grains as revealed by a notched-belly mutant with white-belly. J. Sci. Food Agric.; 2016; 96, pp. 3937-3943. [DOI: https://dx.doi.org/10.1002/jsfa.7793]

53. Zhu, J.; Yu, W.; Zhang, C.; Zhu, Y.; Xu, J.; Li, E.; Gilbert, R.G.; Liu, Q. New insights into amylose and amylopectin biosynthesis in rice endosperm. Carbohyd. Polym.; 2020; 230, 115656. [DOI: https://dx.doi.org/10.1016/j.carbpol.2019.115656]

54. Shi, L. Relationship between Protein Content of Milled Rice and Rice Quality Change. Master’s Thesis; Yangzhou University: Yangzhou, China, 2017.

55. Li, D.D.; Zhen, G.P.; Zhen, Y.; Cai, Y.S.; Zhao, Y.; Pan, S.J.; Huang, C.L.; Wang, L.; Jiang, Y.W.; Zhong, W.J. et al. Relationship between chalkiness index and other quality traits in japonica rice. Jiangsu Agric. Sci.; 2015; 43, pp. 70-72.

56. Chun, A.; Song, J.; Kim, K.; Lee, H. Quality of Head and Chalky Rice and Deterioration of Eating Quality by Chalky Rice. J. Crop Sci. Biotechnol.; 2009; 12, pp. 235-240. [DOI: https://dx.doi.org/10.1007/s12892-009-0142-4]

57. Tong, H. Variety Difference of Rice Quality and Its Relationship with Amylase and Protein Components. Master’s Thesis; Hunan Agricultural University: Changsha, China, 2014.

58. Wang, D.Y.; Zhang, X.F.; Zhu, Z.W.; Chen, N.; Min, J. Correlation analysis of quality traits in edible rice. J. Crops; 2005; 31, pp. 1086-1091.

59. Liu, Y. Study on Physicochemical Basis of Starch and Cytochemical Characteristics of Grain Filling in Rice Quality Difference. Master’s Thesis; Zhejiang University: Hangzhou, China, 2006.

60. Tang, S.; Chen, W.; Liu, W.; Zhou, Q.; Zhang, H.; Ding, S.W.Y. Open-field warming regulates the morphological structure, protein synthesis of grain and affects the appearance quality of rice. J. Cereal Sci.; 2018; 84, pp. 20-29. [DOI: https://dx.doi.org/10.1016/j.jcs.2018.09.013]