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We would like to acknowledge funding from Ministry of Economy and Competitiveness, Spain (RTI2018-097613-BI00 to C.Z., PGC2018-097655-B-I00 to P.C., and AGL2017-85377-R to T.C.); Generalitat de Catalunya Grant 2017 SGR 828 to the Agricultural Biotechnology and Bioeconomy Unit; and the European Union Framework Program DISCO (from discovery to final products: a next-generation pipeline for the sustainable generation of high-value plant products; Project 613513) to P.D.F.

Summary Resistant starch (RS) is a special kind of starch with beneficial effects on obesity, type 2 diabetes and other chronic complications. Breeding high‐RS rice varieties is considered a valuable way to improve public health. However, most rice cultivars only contain an RS level lower than 2% in cooked rice, and cloning of RS genes is critical to improve RS levels in rice. The loss of function of Starch Synthases IIIa (SSIIIa) and SSIIIb, two amylopectin biosynthetic genes, could elevate RS levels up to 10%. Here, we performed a systematic genetic study of 14 amylopectin biosynthetic genes in the ssIIIa ssIIIb double mutant via genome editing, and investigated their effects on RS formation, the eating quality and grain yield. The results showed that deficiency in SSIIa, SSIVb or ISA2 under the ssIIIa ssIIIb background could each elevate RS content to above 14%, and the quadruple mutants of sbeI sbeIIb ssIIIa ssIIIb and sbeI ssIVb ssIIIa ssIIIb could further increase RS levels to over 18%. Furthermore, the eating quality of cooked rice and grain yield decreased along with the elevated RS contents, showing a trade‐off among these traits. In these mutants, ssIIIa ssIIIb showed the balanced performance of RS and grain yield. This study provides insights into RS biosynthesis with a series of RS genes in the amylopectin biosynthesis pathway and practical strategy to breed high‐RS rice varieties with balanced performance.

A core set of genes involved in starch synthesis has been defined by genetic studies, but the complexity of starch biosynthesis has frustrated attempts to elucidate the precise functional roles of the enzymes encoded. The chain-length distribution (CLD) of amylopectin in cereal endosperm is modeled here on the basis that the CLD is produced by concerted actions of three enzyme types: starch synthases, branching and debranching enzymes, including their respective isoforms. The model, together with fitting to experiment, provides four key insights. (1) To generate crystalline starch, defined restrictions on particular ratios of enzymatic activities apply. (2) An independent confirmation of the conclusion, previously reached solely from genetic studies, of the absolute requirement for debranching enzyme in crystalline amylopectin synthesis. (3) The model provides a mechanistic basis for understanding how successive arrays of crystalline lamellae are formed, based on the identification of two independent types of long amylopectin chains, one type remaining in the amorphous lamella, while the other propagates into, and is integral to the formation of, an adjacent crystalline lamella. (4) The model provides a means by which a small number of key parameters defining the core enzymatic activities can be derived from the amylopectin CLD, providing the basis for focusing studies on the enzymatic requirements for generating starches of a particular structure. The modeling approach provides both a new tool to accelerate efforts to understand granular starch biosynthesis and a basis for focusing efforts to manipulate starch structure and functionality using a series of testable predictions based on a robust mechanistic framework.

Amylopectin, the primary form of starch in plant leaves, seeds and tubers, features a tree-like architecture with branched glucose chains. Excess branches result in the formation of soluble phytoglycogen instead of starch granules. In higher plants and green algae, the debranching enzyme isoamylase ISA1 forms either homomultimer or hetero-multimer with ISA2 to facilitate branch trimming and starch granule formation, but the molecular basis remains largely unknown. In this study, we reconstitute the rice OsISA1-ISA2 complex in vitro and determine the cryo-EM structures of the OsISA1 homodimer, as well as the malto-oligosaccharide (MOS)-free and MOS-bound OsISA1-ISA2 heterocomplex. The OsISA1 dimer shows a tail-to-tail rod-like architecture, whereas the OsISA1-ISA2 complex mainly exhibits as a trimer, with OsISA2 flanking on the N-terminal segments of the dimeric OsISA1. Combined with comprehensive biochemical analyses, these structural data elucidate the organization of the ISA1-ISA2 heterocomplex in higher plants and demonstrate how ISA1 and ISA2 cooperate during amylopectin biosynthesis. In plants and algae, isoamylases drive phytoglycogen-to-amylopectin conversion. Here, the authors show that ISA1 and ISA2 form a heterocomplex that coordinately trims glucan branches to promote starch granule formation, defining a key step in starch biosynthesis.

Starch, a plant-derived insoluble carbohydrate composed of glucose polymers, is the principal carbohydrate in our diet and a valuable raw material for industry. The properties of starch depend on the arrangement of glucose units within the constituent polymers. However, key aspects of starch structure and the underlying biosynthetic processes are not well understood, limiting progress towards targeted improvement of our starch crops. In particular, the major component of starch, amylopectin, has a complex three-dimensional, branched architecture. This architecture stems from the combined actions of a multitude of enzymes, each having broad specificities that are difficult to capture experimentally. In this review, we reflect on experimental approaches and limitations to decipher the enzymes’ specificities and explore possibilities for in silico simulations of these activities. We believe that the synergy between experimentation and simulation is needed for the correct interpretation of experimental data and holds the potential to greatly advance our understanding of the overall starch biosynthetic process. We furthermore propose that the formation of glucan secondary structures, concomitant with its synthesis, is a previously overlooked factor that directly affects amylopectin architecture through its impact on enzyme function.

Four starch synthase I (SSI)-deficient rice (Oryza sativa) mutant lines were generated using retrotransposon Tos17 insertion. The mutants exhibited different levels of SSI activities and produced significantly lower amounts of SSI protein ranging from 0% to 20% of the wild type. The mutant endosperm amylopectin showed a decrease in chains with degree of polymerization (DP) 8 to 12 and an increase in chains with DP 6 to 7 and DP 16 to 19. The degree of change in amylopectin chain-length distribution was positively correlated with the extent of decrease in SSI activity in the mutants. The structural changes in the amylopectin increased the gelatinization temperature of endosperm starch. Chain-length analysis of amylopectin in the SSI band excised from native-polyacrylamide gel electrophoresis/SS activity staining gel showed that SSI preferentially synthesized DP 7 to 11 chains by elongating DP 4 to 7 short chains of glycogen or amylopectin. These results show that SSI distinctly generates DP 8 to 12 chains from short DP 6 to 7 chains emerging from the branch point in the A or B₁ chain of amylopectin. SSI seemingly functions from the very early through the late stage of endosperm development. Yet, the complete absence of SSI, despite being a major SS isozyme in the developing endosperm, had no effect on the size and shape of seeds and starch granules and the crystallinity of endosperm starch, suggesting that other SS enzymes are probably capable of partly compensating SSI function. In summary, this study strongly suggested that amylopectin chains are synthesized by the coordinated actions of SSI, SSIIa, and SSIIIa isoforms.

In maize, opaque2 ( o2 ) and opaque 16 ( o16 ) alleles can increase lysine content, while the waxy ( wx ) gene can enhance the amylopectin content of grains. In our study, o2 and o16 alleles were backcrossed into waxy maize line ( wxwx ). The o2o2o16o16wxwx lines had amylopectin contents similar to those of waxy line. Their nutritional value was better than waxy line, but the mechanism by which the o2 and o16 alleles increased the lysine content of waxy maize remained unclear. The o2o2o16o16wxwx lines and their parents on kernels (18th day after pollination) were subjected to RNA sequencing (RNA-Seq). The RNA-Seq analysis revealed 272 differentially expressed genes (DEGs). Functional analyses revealed that these DEGs were mainly related to biomass metabolism. Among them, in o2o2o16o16wxwx lines, 15 genes encoding α-zein were down-regulated, which resulted in the reduction of α-zein synthesis and increased lysine content; lkr/sdh1 and Zm00001d020984.1 genes involved in the lysine degradation pathway were down-regulated, thereby inhibited lysine degradation; sh2 , bt2 and ae1 genes involved in starch metabolism were upregulated, leaded to wrinkling kernel and farinaceous endosperm. Our transcriptional-level identification of key genes responsible for increased grain lysine content and farinaceous endosperm formation following introgression of o2 and o16 alleles should promote molecular breeding for maize quality.

Starch synthase (SS) and branching enzyme (BE) establish the two glycosidic linkages existing in starch. Both enzymes exist as several isoforms. Enzymes derived from several species were studied extensively both in vivo and in vitro over the last years, however, analyses of a functional interaction of SS and BE isoforms are missing so far. Here, we present data from in vitro studies including both interaction of leaf derived and heterologously expressed SS and BE isoforms. We found that SSI activity in native PAGE without addition of glucans was dependent on at least one of the two BE isoforms active in Arabidopsis leaves. This interaction is most likely not based on a physical association of the enzymes, as demonstrated by immunodetection and native PAGE mobility analysis of SSI, BE2, and BE3. The glucans formed by the action of SSI/BEs were analysed using leaf protein extracts from wild type and be single mutants (Atbe2 and Atbe3 mutant lines) and by different combinations of recombinant proteins. Chain length distribution (CLD) patterns of the formed glucans were irrespective of SSI and BE isoforms origin and still independent of assay conditions. Furthermore, we show that all SS isoforms (SSI-SSIV) were able to interact with BEs and form branched glucans. However, only SSI/BEs generated a polymodal distribution of glucans which was similar to CLD pattern detected in amylopectin of Arabidopsis leaf starch. We discuss the impact of the SSI/BEs interplay for the CLD pattern of amylopectin.

The composition of amylopectin is the determinant of rice eating quality under certain threshold of protein content and the ratio of amylose and amylopectin. In molecular biology level, the fine structure of amylopectin is determined by relative activities of starch branching enzyme (SBE), granule-bound starch synthase (GBSS), and soluble starch synthase (SSS) in rice grain under the same ADP-Glucose level. But the underlying mechanism of eating quality in molecular biology level remains unclear. This paper reports the differences on major parameters such as SNP and insertion-deletion sites, RNA expressions, and enzyme activities associated with eating quality of japonica varieties. Eight japonica rice varieties with significant differences in various eating quality parameters such as palatability and protein content were used in this experiment. Association analysis between nucleotide polymorphism and eating quality showed that S12 and S13 loci in SBE1, S55 in SSS1, S58 in SSS2A were significantly associated with apparent amylose content, alkali digestion value, setback viscosity, consistency viscosity, pasting temperature, which explained most of the variation in apparent amylose content, setback viscosity, and consistency viscosity; and explained almost all variations in alkali digestion value and pasting temperature. Thirty-five SNPs and insertion-deletions from SBE1, SBE3, GBSS1, SSS1, and SSS2A differentiated high or intermediate palatability rice varieties from low palatability rice varieties. Correlation analysis between enzyme activities and eating quality properties revealed that SBE25 and SSS15/W15 were positively correlated with palatability, whereas GBSS10 and GBSS15 were negatively correlated. Gene expressions showed that SBE1 and SBE3 expressions in high palatability varieties tended to be higher than middle and low palatability varieties. Collectively, SBE1, SBE3, SSS1, and SSS2A, especially SBE1 and SBE3 could improve eating quality, but GBSS1 decreased eating quality. The results indicated the possibility of developing high palatability cultivars through modification of key genes related to japonica rice eating quality formation in starch biosynthesis.

To elucidate the role of SSIIIa during starch synthesis in rice (Oryza sativa L.) endosperm, we characterized null mutants of this gene, generated by T-DNA insertions. Scanning electron microscope (SEM) analysis revealed that the starch granules in these mutants are smaller and rounder compared with the wild type controls, and that the mutant endosperm is characterized by a loosely packed central portion exhibiting a floury-like phenotype. Hence, the OsSSIIIa (Oryza sativa SSIIIa) mutations are referred to as white-core floury endosperm 5-1 (flo5-1) and flo5-2. Based upon their X-ray diffraction patterns, the crystallinity of the starch in the flo5 mutant endosperm is decreased compared with wild type. Through determination of the chain-length distribution of the mutant endosperm starch, we found that flo5-1 and flo5-2 mutants have reduced the content of long chains with degree of polymerization (DP) 30 or greater compared with the controls. This suggests that OsSSIIIa/Flo5 plays an important role in generating relatively long chains in rice endosperm. In addition, DP 6 to 8 and DP 16 to 20 appeared to be reduced in endosperm starch of flo5-1 and flo5-2, whereas DP 9 to 15 and DP 22 to 29 were increased in these mutants. By the use of differential scanning calorimetry (DSC), the gelatinization temperatures of endosperm starch were found to be 1-5°C lower than those of the control. We propose a distinct role for OsSSIIIa/Flo5 and the coordinated action of other SS isoforms during starch synthesis in the seed endosperm of rice.