Correction: Ying et al. Dynamic Change in Starch Biosynthetic Enzymes Complexes during Grain-Filling Stages in BEIIb Active and Deficient Rice. Int. J. Mol. Sci. 2022, 23, 10714.

In the original publication [...].

Relative importance of branching enzyme isoforms in determining starch fine structure and physicochemical properties of indica rice.

KEY MESSAGE: Down-regulation of starch branching enzymes alters fine structure and starch properties, especially the B-type crystalline pattern and extremely high amylose content identified in the BEIIb-deficiency mutant in the indica rice. The relative importance of the starch branching enzymes in determining the molecular fine structure and starch functional properties were uncovered in this study. An indica rice, Guangluai 4 with high amylose content (AC) and high gelatinization temperature (GT) was used to generate the clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein-9 (Cas9) knockout lines. Five mutant lines were identified including be1-1, be1-2, be2a-1, be2a-2 and be2b-1, and analysis of western blot showed the CRISPR/Cas9 system was successful in inducing mutations in the targeted genes. AC of be2b-1 (34.1%) was greater than that of wild type (WT) (27.4%) and other mutants. Mutations of either BEI or BEIIa did not alter the starch crystallite pattern (A-type). The BEIIb deficiency caused an opaque endosperm phenotype, changed the crystallite pattern from A- to B-type, and dramatically increased the degree of ordered structure, the relative proportion of amylose chains and intermediate to long amylopectin chains, average chain length of amylopectin molecules as well as GT. The BEIIa deficiency had no effect on the proportion of amylose chains, the length of amylopectin intermediate-long chains, conclusion temperature and enthalpy of gelatinization. Down-regulation of BEI increased the proportion of shortest amylopectin chains (fa) but decreased the proportion of long amylopectin chains (fb2 and fb3), leading to a lower GT. It is concluded that the relative importance in determining starch fine structures and functionality was in the order of BEIIb > BEI > BEIIa. Our results provide new information for utilizations of BE-deficient mutants in rice quality breeding.

Functional Interactions between Enzymes Involved in Amylose and Amylopectin Biosynthesis in Rice Based on Mathematical Models.

Starch biosynthesis is controlled by multiple enzymes, including granule-bound starch synthase I (GBSSI), soluble starch synthases (SSs), branching enzymes (BEs), and debranching enzymes (DBEs). Although the role of individual isoforms has been primarily elucidated, the precise information about how they work together in the synthesis of specific amylose and amylopectin chains is still unclear. In this study, starch molecular chain-length distributions (CLDs) of five rice varieties with different amylose contents were measured by fluorophore-assisted carbohydrate electrophoresis and size-exclusion chromatography and fitted with two mathematical models, and the protein abundance of 11 starch synthesis-related enzymes was measured by western blotting. The correlation between model fitting parameters of amylose and amylopectin CLDs demonstrated that amylose and amylopectin syntheses are closely dependent. GBSSI could interact with BEI, BEIIb, SSIIa, SSIVb, ISA1, PUL, and PHO1 to synthesize the amylopectin intermediate and long chains as well as amylose chains. In addition, the interaction among SSIVb and SSI, SSIIa, BEI, BEIIb, ISA1, and PUL possibly suggests that SSIVb assists them to synthesize the amylopectin chains. The results can help understand the mechanisms about the functional interaction of different enzyme isoforms in starch biosynthesis.

Dynamic Change in Starch Biosynthetic Enzymes Complexes during Grain-Filling Stages in BEIIb Active and Deficient Rice.

Starch is the predominant reserve in rice (Oryza sativa L.) endosperm, which is synthesized by the coordinated efforts of a series of starch biosynthetic-related enzymes in the form of a multiple enzyme complex. Whether the enzyme complex changes during seed development is not fully understood. Here, we investigated the dynamic change in multi-protein complexes in an indica rice variety IR36 (wild type, WT) and its BEIIb-deficient mutant (be2b) at different developmental stages. Gel permeation chromatography (GPC) and Western blotting analysis of soluble protein fractions revealed most of the enzymes except for SSIVb were eluted in smaller molecular weight fractions at the early developing stage and were transferred to higher molecular weight fractions at the later stage in both WT and be2b. Accordingly, protein interactions were enhanced during seed development as demonstrated by co-immunoprecipitation analysis, suggesting that the enzymes were recruited to form larger protein complexes during starch biosynthesis. The converse elution pattern from GPC of SSIVb may be attributed to its vital role in the initiation step of starch synthesis. The number of protein complexes was markedly decreased in be2b at all development stages. Although SSIVb could partially compensate for the role of BEIIb in protein complex formation, it was hard to form a larger protein complex containing over five proteins in be2b. In addition, other proteins such as PPDKA and PPDKB were possibly present in the multi-enzyme complexes by proteomic analyses of high molecular weight fractions separated from GPC. Two putative protein kinases were found to be potentially associated with starch biosynthetic enzymes. Collectively, our findings unraveled a dynamic change in the protein complex during seed development, and potential roles of BEIIb in starch biosynthesis via various protein complex formations, which enables a deeper understanding of the complex mechanism of starch biosynthesis in rice.

Proteomics and Post-Translational Modifications of Starch Biosynthesis-Related Proteins in Developing Seeds of Rice.

Rice (Oryza sativa L.) is a foremost staple food for approximately half the world's population. The components of rice starch, amylose, and amylopectin are synthesized by a series of enzymes, which are responsible for rice starch properties and functionality, and then affect rice cooking and eating quality. Recently, proteomics technology has been applied to the establishment of the differentially expressed starch biosynthesis-related proteins and the identification of posttranslational modifications (PTMs) target starch biosynthesis proteins as well. It is necessary to summarize the recent studies in proteomics and PTMs in rice endosperm to deepen our understanding of starch biosynthesis protein expression and regulation, which will provide useful information to rice breeding programs and industrial starch applications. The review provides a comprehensive summary of proteins and PTMs involved in starch biosynthesis based on proteomic studies of rice developing seeds. Starch biosynthesis proteins in rice seeds were differentially expressed in the developing seeds at different developmental stages. All the proteins involving in starch biosynthesis were identified using proteomics methods. Most starch biosynthesis-related proteins are basically increased at 6-20 days after flowering (DAF) and decreased upon the high-temperature conditions. A total of 10, 14, 2, 17, and 7 starch biosynthesis related proteins were identified to be targeted by phosphorylation, lysine acetylation, succinylation, lysine 2-hydroxyisobutyrylation, and malonylation, respectively. The phosphoglucomutase is commonly targeted by five PTMs types. Research on the function of phosphorylation in multiple enzyme complex formation in endosperm starch biosynthesis is underway, while the functions of other PTMs in starch biosynthesis are necessary to be conducted in the near future.

Physicochemical characteristics of tea seed starches from twenty-five cultivars.

The physicochemical features of starches separated from tea seeds of 25 cultivars were analyzed. The distinct characteristic of tea seed starches was that they had high apparent amylose content (AAC, 28.94-39.91 %) and resistant starch contents (4.64-8.24 %), suggesting that tea starch can be used for production of low glycemic index food. One cultivar (T12) had smallest breakdown (74.2 RVU) and highest gel hardness, indicating it performed stably during shear thinning, resulting in a firm texture. Another cultivar (T25) had a peak viscosity of 417.6 RVU, a large breakdown and small setback, suggesting a low tendency for retrogradation. There was a range of 61.6 °C to 77.5 °C for the peak gelatinization temperature and 0.163 to 0.390 for the flow behavior index values. These parameters could serve for selecting suitable starches with minor differences in physicochemical properties for food use. Correlation analysis indicated that AAC is a key factor determining starch retrogradation properties. The broad genetic diversity in the tea seed starch physicochemical features provided potentially versatile applications in the food industry. The results gained from the present study contribute to a better understanding of tea seed starch quality, and encourage its application in many value-added food products.

Identification of a new allele of soluble starch synthase IIIa involved in the elongation of amylopectin long chains in a chalky rice mutant.

A chalky endosperm mutant (GM03) induced from an indica rice GLA4 was used to investigate the functional gene in starch biosynthesis. Bulked segregant analysis and sanger sequencing determined that a novel mutation in soluble starch synthase IIIa (SSIIIa) is responsible for the chalky phenotype in GM03. Complementary test by transforming the active SSIIIa gene driven by its native promoter to GM03 recovered the phenotype to its wildtype. The expression of SSIIIa was significantly decreased, while SSIIIa protein was not detected in GM03. The mutation of SSIIIa led to increased expression of most of starch synthesis related genes and elevated the levels of most of proteins in GM03. The CRISPR/Cas9 technology was used for targeted disruption of SSIIIa, and the mutant lines exhibited chalky endosperm which phenocopied the GM03. Additionally, the starch fine structure in the knockout mutant lines ss3a-1 and ss3a-2 was similar with the GM03, which showed increased amylose content, higher proportions of B1 and B2 chains, much lower proportions of B3 chains and decreased degree of crystallinity, leading to altered thermal properties with lower gelatinization temperature and enthalpy. Collectively, these results suggested that SSIIIa plays an important role in starch synthesis by elongating amylopectin long chains in rice.

Diurnal changes in starch molecular structures and expression profiles of starch biosynthesis enzymes in rice developing seeds.

The diurnal changes in the expression profiles of starch synthesis related enzymes (SSREs) has been previously studied in transitory starches, while its influences on storage starch molecular structures in the rice endosperm during seed development have not been elucidated. In this study, the changes in the transcript levels of starch synthesis related genes (SSRGs), the protein abundances and enzyme activities of SSREs as well as starch molecular structures in rice endosperm at 10 days after flowering (DAF) over the diurnal cycle were analyzed. It was found that the expression profiles of SSRG and the protein contents of SSREs displayed different diurnal patterns between two indica rice varieties with medium- and high-amylose content (AC), respectively. The expression levels of SSRGs were higher in the light time, and most SSREs also accumulated during this period except debranching enzymes. Amylose synthesis displayed distinct diurnal patterns in two rice varieties, which is attributed to the diurnal changes in the protein content of granule-bound starch synthase I (GBSSI), but amylopectin chain-length distributions (CLDs) remained unaltered due to its vast numbers of branches. The results provide the first step to understand the roles of each enzyme isoform involved in starch synthesis in response to diurnal regulation in rice endosperm.

Starch fine structure and functional properties during seed development in BEIIb active and deficient rice.

Loss of starch branching enzyme IIb (BEIIb) leads to altered starch structure and increased amylose content. The changes in starch fine structure and function during seed development were investigated as a result from differentially expressed genes between wildtype (WT) and be2b rice. The expression patterns of all starch synthesis related genes except the AGPS1 were altered in be2b. From five to 15 days after flowering (DAF), the amylose content and proportion of A chains of amylopectin increased, while those of B2, B3 chains and average chain length declined in both WT and be2b. The mutant had a C-type crystalline pattern and a higher relative crystallinity (RC) at five and 10 DAF, which was transferred to a B-type and a lower RC at 15 DAF in be2b, while the WT had A-type starch at all developmental stages. A possible model for amylose and amylopectin structure in WT and be2b was proposed.

The role of different Wx and BEIIb allele combinations on fine structures and functional properties of indica rice starches.

This study examined the effects of the combinations of Waxy (Wx) and starch branching enzyme IIb (BEIIb) alleles on starch fine structure and functional properties in indica rice cultivars. The results showed that be2b mutant starches with BEIIb deficiency had higher amylose content, shorter amylose long chains, a higher proportion of amylopectin long chains and molecular order, but a lower proportion of amylopectin short chains and relative crystallinity, resulting in higher gelatinization temperature but lower enthalpy and paste viscosity. Compared with the rice lines carrying Wxb allele, Wxa allele contributed to relatively higher amylose content, longer amylopectin chains, less short-range ordered structure and lower relative crystallinity, leading to a little lower gelatinization enthalpy. This study provides new insight into structure-function relations among rice lines with different allele combinations of starch synthesis related genes, which is a useful strategy for rice quality breeding.

Combined Effects of Different Alleles of FLO2, Wx and SSIIa on the Cooking and Eating Quality of Rice.

The improvement of the cooking and eating quality (CEQ) of rice is one of the major objectives of current rice-breeding programs. A few major genes such as Waxy (Wx) and starch synthase IIa (SSIIa) have been successfully applied in molecular breeding. However, their interactive effects on CEQ have not been fully understood. In this study, a recombinant inbred line (RIL) population was constructed by crossing the white-core mutant GM645 with the transparent phenotype of the japonica rice variety Tainung 67 (TN67). GM645 and TN67 contain different alleles of FLOURY ENDOSPERM2 (FLO2), Wx, and SSIIa. The effects of different allele combinations of FLO2, Wx, and SSIIa on the CEQ of rice were investigated. The inbred lines with the mutation allele flo2 had a significantly lower apparent amylose content (AAC), viscosity characteristics except for setback (SB), and gel texture properties compared to those lines with the FLO2 allele. The allelic combination of FLO2 and Wx significantly affected the AAC, breakdown (BD), and gel textural properties, which could explain most of the variations in those rice quality traits that were correlated with AAC. The allelic combination of FLO2 and SSIIa significantly affected the hot paste viscosity (HPV) and pasting temperature (PT). The Wx × SSIIa interaction had a significant effect on the PT. The interaction of FLO2, Wx and SSIIa significantly affected the AAC, cold paste viscosity (CPV), PT, and consistency viscosity (CS). These results highlight the important roles of these quality-related genes in regulating the CEQ of rice and provide new clues for rice-quality improvement by marker-assisted selection.

Relationships among starch biosynthesizing protein content, fine structure and functionality in rice.

The underlying mechanisms behind the genetics, structure and functionality relations in starches from various origins have not been fully understood. For better control of the genotypic background, rice starches from four chalky mutants and their parent were employed to investigate the above relations, and some interesting relations were revealed. GBSSI and SSIIIa were shown to affect the height of amylose in the debranched starch fraction (hAM) which reflects true amylose content (TAC), and then hAM was correlated with the AAC, RS, HD, COH, To, ΔHg, ΔHr, and R%. GBSSI also affected the average chain length (X)¯ of amylopectin, which was associated with the AAC, TAC, RS, HPV, HD, COH, ΔHg, ΔHr, and R%. The SSI, SSIIa and SSIIIa affected the amylose size (Rh,AM), which was correlated with the TAC, AAC, RS, HD, COH, To and Tp. Furthermore, both SSIIa and PUL affected the XAP2, and XAP2 was correlated with the To.

Gelatinization, pasting and retrogradation properties and molecular fine structure of starches from seven cassava cultivars.

Genetic diversity in the physicochemical properties and fine structures of seven cassava starches samples was studied. The apparent amylose content ranged from 24.8 to 27.6%. The whole branched starches showed significant differences in average hydrodynamic radius, ranging from 53.35 to 58.45 nm, while debranched starch exhibited differences in degrees of polymerization and height of both amylose and amylopectin peaks. The molecular size of amylose and amylopectin was positively correlated. The amount of short chains fa (6 ≤ X ≤ 12) and fb1 (13 ≤ X ≤ 24) had significant differences among the cultivars. Structure-function relation analysis indicated that the CPV and SB were mainly determined by amylopectin fine structures, BD, PTi and Tp and retrogradation properties were mainly determined by the amylose fine structure, while PTe and To were mainly affected by both amylose and amylopectin fine structures. The current findings will be helpful to improve the understanding cassava starch quality for use in industrial starch applications.

Physicochemical properties and digestibility of endosperm starches in four indica rice mutants.

Structural, physicochemical characteristics and digestibility of endosperm starches from four mutants (GM01, 03-05) and their parent Guangluai 4 (GLA4) were characterized. GM05 had a little higher apparent amylose content (AAC) and showed little difference in starch and digestion properties from GLA4. However, GM03 and GM04 with increased amount of chalkiness and partial translucent region in the endosperm displayed a distinct starch and digestion properties, which had the RS of 7.9 and 8.4%, respectively. GM03 and GM04 had higher AAC (33-35%), lower degree of crystallinity, lower gelatinization temperature and pasting viscosities, and more amount of B1 (DP 12-24) and B2 (DP 25-36) chains and less amount of B3 chains (DP ≥ 37) in amylopectin. AAC and the amount of B1 chains had positive correlation with RS, but the amount of B3 chains had negative correlation with RS. The results of this study may be applied to design RS by selecting rice germplasm with high AAC and high amount of B1 chains (DP 12-24) of amylopectin.