A comprehensive overview of omics-based approaches to enhance biotic and abiotic stress tolerance in sweet potato.

Biotic and abiotic stresses negatively affect the yield and overall plant developmental process, thus causing substantial losses in global sweet potato production. To cope with stresses, sweet potato has evolved numerous strategies to tackle ever-changing surroundings and biological and environmental conditions. The invention of modern sequencing technology and the latest data processing and analysis instruments has paved the way to integrate biological information from different approaches and helps to understand plant system biology more precisely. The advancement in omics technologies has accumulated and provided a great source of information at all levels (genome, transcript, protein, and metabolite) under stressful conditions. These latest molecular tools facilitate us to understand better the plant's responses to stress signaling and help to process/integrate the biological information encoded within the biological system of plants. This review briefly addresses utilizing the latest omics strategies for deciphering the adaptive mechanisms for sweet potatoes' biotic and abiotic stress tolerance via functional genomics, transcriptomics, proteomics, and metabolomics. This information also provides a powerful reference to understand the complex, well-coordinated stress signaling genetic regulatory networks and better comprehend the plant phenotypic responses at the cellular/molecular level under various environmental stimuli, thus accelerating the design of stress-resilient sweet potato via the latest genetic engineering approaches.

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 [...].

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.

Effects of single and dual modifications through electron beam irradiation and hydroxypropylation on physicochemical properties of potato and corn starches.

In this study, electron beam irradiation (EBI; 2, 4, 6, 8 and 10 kGy), hydroxypropylation (HP) and dual modification of EBI-HP were applied to modify corn and potato starches. The results showed that the molar substitution (MS) of EBI-HP modified corn and potato starches were in the range of 0.060-0.087 and 0.080-0.124, respectively. After modifications, amylose content of corn (30.0 %) and potato (31.2 %) starches were declined to 24.2-28.1 % and 26.1-29.5 %, respectively, and relative crystallinity was reduced from 35.5 to 30.0 % for corn and 34.1 to 20.2 % for potato. Pasting properties decreased significantly in both starch sources with increasing irradiation dose. EBI decreased springiness, enthalpy of retrograded starch (ΔHr) and percentage of retrogradation (R%) on corn starches, which were different from those effects observed on potato starches. Meanwhile, HP increased peak viscosity up to 312.6 RVU and 1359.3 RVU for corn and potato starches, respectively. Moreover, EBI-HP was highly responsible for the decreases in the textural, gelatinization and retrogradation properties and relative crystallinity in both corn and potato starches. These results enhance the understanding of starch functionality modified by using both physical and chemical methods, and provide further insights on food and non-food applications.

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.

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.

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.

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.

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.

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.

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.

Identification and expression of genes in response to cassava bacterial blight infection.

Cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (or XAM) is a serious disease of cassava (Manihot esculenta Crantz). In this study, quantitative trait loci (QTL) associated with CBB infection were identified in the F1 progenies of a cross between the "Huay Bong 60" and "Hanatee" cassava cultivars. The phenotype of disease severity was observed at 7, 10, and 12 days after inoculation (DAI). A total of 12 QTL were identified, of which 5, 6, and 1 were detected in 7, 10, and 12 DAI samples, respectively. Among all identified QTL, CBB14_10dai_1, CBB14_10dai_2, and CBB14_12dai showed the most significant (P < 0.0001) associations with CBB infection, and explained 21.3, 13.8, and 26.5% of phenotypic variation, respectively. Genes underlying the QTL were identified and their expression was investigated in resistant and susceptible cassava plants by real-time quantitative RT-PCR. The results identified candidate genes that showed significant differences in expression between resistant and susceptible lines, including brassinosteroid insensitive 1-associated receptor kinase 1-related (Manes.04G059100), cyclic nucleotide-gated ion channel 2 (Manes.02G051100), and autophagy-related protein 8a-related (Manes.17G026600) at 7 DAI, and regulator of nonsense transcripts 1 homolog (Manes.17G021900) at both 7 and 12 DAI. The expression pattern of all genes showed higher levels in resistant (B82, B32, B20, and B70) as compared to susceptible (HB60, B100, B95, and B47) plants. Overall, this study has identified QTL and markers linked to CBB infection trait, and identified candidate genes involved in CBB resistance. This information will be of use for better understanding defense mechanisms in cassava to bacterial blight disease.