Rice LIKE EARLY STARVATION1 cooperates with FLOURY ENDOSPERM6 to modulate starch biosynthesis and endosperm development.

In cereal grains, starch is synthesized by the concerted actions of multiple enzymes on the surface of starch granules within the amyloplast. However, little is known about how starch-synthesizing enzymes access starch granules, especially for amylopectin biosynthesis. Here, we show that the rice (Oryza sativa) floury endosperm9 (flo9) mutant is defective in amylopectin biosynthesis, leading to grains exhibiting a floury endosperm with a hollow core. Molecular cloning revealed that FLO9 encodes a plant-specific protein homologous to Arabidopsis (Arabidopsis thaliana) LIKE EARLY STARVATION1 (LESV). Unlike Arabidopsis LESV, which is involved in starch metabolism in leaves, OsLESV is required for starch granule initiation in the endosperm. OsLESV can directly bind to starch by its C-terminal tryptophan (Trp)-rich region. Cellular and biochemical evidence suggests that OsLESV interacts with the starch-binding protein FLO6, and loss-of-function mutations of either gene impair ISOAMYLASE1 (ISA1) targeting to starch granules. Genetically, OsLESV acts synergistically with FLO6 to regulate starch biosynthesis and endosperm development. Together, our results identify OsLESV-FLO6 as a non-enzymatic molecular module responsible for ISA1 localization on starch granules, and present a target gene for use in biotechnology to control starch content and composition in rice endosperm.

LIKE EARLY STARVATION 1 interacts with amylopectin during starch biosynthesis.

Starch is the major energy storage compound in plants. Both transient starch and long-lasting storage starch accumulate in the form of insoluble, partly crystalline granules. The structure of these granules is related to the structure of the branched polymer amylopectin: linear chains of glucose units organized in double helices that align to form semicrystalline lamellae, with branching points located in amorphous regions between them. EARLY STARVATION 1 (ESV1) and LIKE EARLY STARVATION 1 (LESV) proteins are involved in the maintenance of starch granule structure and in the phase transition of amylopectin, respectively, in Arabidopsis (Arabidopsis thaliana). These proteins contain a conserved tryptophan-rich C-terminal domain folded into an antiparallel ß-sheet, likely responsible for binding of the proteins to starch, and different N-terminal domains whose structure and function are unknown. In this work, we combined biochemical and biophysical approaches to analyze the structures of LESV and ESV1 and their interactions with the different starch polyglucans. We determined that both proteins interact with amylopectin but not with amylose and that only LESV is capable of interacting with amylopectin during starch biosynthesis. While the C-terminal domain interacts with amylopectin in its semicrystalline form, the N-terminal domain of LESV undergoes induced conformational changes that are probably involved in its specific function of mediating glucan phase transition. These results clarify the specific mechanism of action of these 2 proteins in the biosynthesis of starch granules.

Creating a zero amylose barley with high soluble sugar content by genome editing.

Amylose biosynthesis is strictly associated with granule-bound starch synthase I (GBSSI) encoded by the Waxy gene. Mutagenesis of single bases in the Waxy gene, which induced by CRISPR/Cas9 genome editing, caused absence of intact GBSSI protein in grain of the edited line. The amylose and amylopectin contents of waxy mutants were zero and 31.73%, while those in the wild type were 33.50% and 39.00%, respectively. The absence of GBSSI protein led to increase in soluble sugar content to 37.30% compared with only 10.0% in the wild type. Sucrose and ß-glucan, were 39.16% and 35.40% higher in waxy mutants than in the wild type, respectively. Transcriptome analysis identified differences between the wild type and waxy mutants that could partly explain the reduction in amylose and amylopectin contents and the increase in soluble sugar, sucrose and ß-glucan contents. This waxy flour, which showed lower final viscosity and setback, and higher breakdown, could provide more option for food processing.

Sticking to starch.

Not all starches in the human diet are created equal: "resistant starches" are consolidated aggregates of the α-glucan polysaccharides amylose and amylopectin, which escape digestion by salivary and pancreatic amylases. Upon reaching the large intestine, resistant starches become fodder for members of the human gut microbiota, impacting the metabolism of both the symbionts and the host. In a recent study, Koropatkin et al. provided new molecular insight into how a keystone bacterium in the human gut microbiota adheres to resistant starches as a prelude to their breakdown and fermentation.

Sas20 is a highly flexible starch-binding protein in the Ruminococcus bromii cell-surface amylosome.

Ruminococcus bromii is a keystone species in the human gut that has the rare ability to degrade dietary resistant starch (RS). This bacterium secretes a suite of starch-active proteins that work together within larger complexes called amylosomes that allow R. bromii to bind and degrade RS. Starch adherence system protein 20 (Sas20) is one of the more abundant proteins assembled within amylosomes, but little could be predicted about its molecular features based on amino acid sequence. Here, we performed a structure-function analysis of Sas20 and determined that it features two discrete starch-binding domains separated by a flexible linker. We show that Sas20 domain 1 contains an N-terminal ß-sandwich followed by a cluster of α-helices, and the nonreducing end of maltooligosaccharides can be captured between these structural features. Furthermore, the crystal structure of a close homolog of Sas20 domain 2 revealed a unique bilobed starch-binding groove that targets the helical α1,4-linked glycan chains found in amorphous regions of amylopectin and crystalline regions of amylose. Affinity PAGE and isothermal titration calorimetry demonstrated that both domains bind maltoheptaose and soluble starch with relatively high affinity (Kd ≤ 20 µM) but exhibit limited or no binding to cyclodextrins. Finally, small-angle X-ray scattering analysis of the individual and combined domains support that these structures are highly flexible, which may allow the protein to adopt conformations that enhance its starch-targeting efficiency. Taken together, we conclude that Sas20 binds distinct features within the starch granule, facilitating the ability of R. bromii to hydrolyze dietary RS.

Evaluation of altered starch mutants and identification of candidate genes responsible for starch variation in wheat.

BACKGROUND: Induction of mutation through chemical mutagenesis is a novel approach for preparing diverse germplasm. Introduction of functional alleles in the starch biosynthetic genes help in the improvement of the quality and yield of cereals. RESULTS: In the present study, a set of 350 stable mutant lines were used to evaluate dynamic variation of the total starch contents. A megazyme kits were used for measuring the total starch content, resistant starch, amylose, and amylopectin content. Analysis of variance showed significant variation (p < 0.05) in starch content within the population. Furthermore, two high starch mutants (JE0173 and JE0218) and two low starch mutants (JE0089 and JE0418) were selected for studying different traits. A multiple comparison test showed that significant variation in all physiological and morphological traits, with respect to the parent variety (J411) in 2019-2020 and 2020-2021. The quantitative expression of starch metabolic genes revealed that eleven genes of JE0173 and twelve genes of JE0218 had consistent expression in high starch mutant lines. Similarly, in low starch mutant lines, eleven genes of JE0089 and thirteen genes of JE0418 had consistent expression in all stages of seed development. An additional two candidate genes showed over-expression (PHO1, PUL) in the high starch mutant lines, indicating that other starch metabolic genes may also contribute to the starch biosynthesis. The overexpression of SSII, SSIII and SBEI in JE0173 may be due to presence of missense mutations in these genes and SSI also showed overexpression which may be due to 3-primer_UTR variant. These mutations can affect the other starch related genes and help to increase the starch content in this mutant line (JE0173). CONCLUSIONS: This study screened a large scale of mutant population and identified mutants, could provide useful genetic resources for the study of starch biosynthesis and genetic improvement of wheat in the future. Further study will help to understand new genes which are responsible for the fluctuation of total starch.

The OsNAC24-OsNAP protein complex activates OsGBSSI and OsSBEI expression to fine-tune starch biosynthesis in rice endosperm.

Starch accounts for up to 90% of the dry weight of rice endosperm and is a key determinant of grain quality. Although starch biosynthesis enzymes have been comprehensively studied, transcriptional regulation of starch-synthesis enzyme-coding genes (SECGs) is largely unknown. In this study, we explored the role of a NAC transcription factor, OsNAC24, in regulating starch biosynthesis in rice. OsNAC24 is highly expressed in developing endosperm. The endosperm of osnac24 mutants is normal in appearance as is starch granule morphology, while total starch content, amylose content, chain length distribution of amylopectin and the physicochemical properties of the starch are changed. In addition, the expression of several SECGs was altered in osnac24 mutant plants. OsNAC24 is a transcriptional activator that targets the promoters of six SECGs; OsGBSSI, OsSBEI, OsAGPS2, OsSSI, OsSSIIIa and OsSSIVb. Since both the mRNA and protein abundances of OsGBSSI and OsSBEI were decreased in the mutants, OsNAC24 functions to regulate starch synthesis mainly through OsGBSSI and OsSBEI. Furthermore, OsNAC24 binds to the newly identified motifs TTGACAA, AGAAGA and ACAAGA as well as the core NAC-binding motif CACG. Another NAC family member, OsNAP, interacts with OsNAC24 and coactivates target gene expression. Loss-of-function of OsNAP led to altered expression in all tested SECGs and reduced the starch content. These results demonstrate that the OsNAC24-OsNAP complex plays key roles in fine-tuning starch synthesis in rice endosperm and further suggest that manipulating the OsNAC24-OsNAP complex regulatory network could be a potential strategy for breeding rice cultivars with improved cooking and eating quality.

Insights into the Supramolecular Structure and Degradation Mechanisms of Starch from Different Botanical Sources as Affected by Extrusion-based 3D Printing.

Extrusion-based 3D printing has emerged as the most versatile additive manufacturing technique for the printing of practically any material. However, 3D printing of functional materials often activates thermo-mechanical degradation, which affects the 3D shape quality. Herein, we describe the structural changes of eight different starch sources (normal or waxy) as a consequence of the temperature of an extrusion-based 3D printing system through in-depth characterization of their molecular and structural changes. The combination of size-exclusion chromatography, small-angle X-ray scattering, X-ray diffraction, dynamic viscoelasticity measurements, and in vitro digestion has offered an extensive picture of the structural and biological transformations of starch varieties. Depending on the 3D printing conditions, either gelatinization was attained ("moderate" condition) or single-amylose helix formation was induced ("extreme" condition). The stiff amylopectin crystallites in starch granules were more susceptible to thermo-mechanical degradation compared to flexible amorphous amylose. The crystalline morphology of the starch varieties varied from B-type crystallinity for the starch 3D printing at the "moderate" condition to a mixture of C- and V-type crystallinity regarding the "extreme" condition. The "extreme" condition reduced the viscoelasticity of 3D-printed starches but increased the starch digestibility rate/extent. In contrast, the "moderate" condition increased the viscoelastic moduli, decreasing the starch digestion rate/extent. This was more considerable mainly regarding the waxy starch varieties. Finally, normal starch varieties presented a well-defined shape fidelity, being able to form a stable structure, whereas waxy starches exhibited a non-well-defined structure and were not able to maintain their integrity after printing. The results of this research allow us to monitor the degradability of a variety of starch cultivars to create starch-based 3D structures, in which the local structure can be controlled based on the 3D printing parameters.

Evaluation of pea/rice and amylopectin/chromium as an alternative protein source to improve muscle protein synthesis in rats.

BACKGROUND: A preclinical study reported that the combination of an amylopectin/chromium complex (ACr) of branched-chain amino acids (BCAA) significantly enhanced muscle protein synthesis (MPS). This study was conducted to determine the effects of the addition of ACr complex to a pea/rice (PR) protein on MPS, insulin, muslin levels, and the mTOR pathway in exercised rats. METHODS: Twenty-four rats were divided into three groups: (i) exercise (Ex); (ii) Ex + PR 1:1 blend (0.465 g/kg BW); (iii) Ex + PR + ACr (0.155 g/kg BW). On the day of single-dose administration, after the animals were exercised at 26/m/min for 2 h, the supplement was given by oral gavage. The rats were injected with a bolus dose (250 mg/kg BW, 25 g/L) of deuterium-labeled phenylalanine to determine the protein fractional synthesis rate (FSR) one h after consuming the study product. RESULTS: The combination of PR and ACr enhanced MPS by 42.55% compared to the Ex group, while Ex + PR alone increased MPS by 30.2% over the Ex group (p < 0.0001) in exercised rats. Ex + PR plus ACr significantly enhanced phosphorylation of mTOR and S6K1 (p < 0.0001), and 4E-BP1 (p < 0.001) compared to the Ex (p < 0.0001). PR to ACr also significantly increased insulin and musclin levels (p < 0.0001) in exercised rats. Additionally, compared to Ex + PR alone, Ex + PR + ACr enhanced mTOR (p < 0.0001) and S6K1 (p < 0.0001) levels. CONCLUSION: These data suggested that PR + ACr may provide an alternative to animal proteins for remodeling and repairing muscle by stimulating MPS and mTOR signaling pathways in post-exercised rats. More preclinical and clinical human studies on combining pea/rice and amylopectin/chromium complex are required.

Genetic Engineering of Starch Biosynthesis in Maize Seeds for Efficient Enzymatic Digestion of Starch during Bioethanol Production.

Maize accumulates large amounts of starch in seeds which have been used as food for human and animals. Maize starch is an importantly industrial raw material for bioethanol production. One critical step in bioethanol production is degrading starch to oligosaccharides and glucose by α-amylase and glucoamylase. This step usually requires high temperature and additional equipment, leading to an increased production cost. Currently, there remains a lack of specially designed maize cultivars with optimized starch (amylose and amylopectin) compositions for bioethanol production. We discussed the features of starch granules suitable for efficient enzymatic digestion. Thus far, great advances have been made in molecular characterization of the key proteins involved in starch metabolism in maize seeds. The review explores how these proteins affect starch metabolism pathway, especially in controlling the composition, size and features of starch. We highlight the roles of key enzymes in controlling amylose/amylopectin ratio and granules architecture. Based on current technological process of bioethanol production using maize starch, we propose that several key enzymes can be modified in abundance or activities via genetic engineering to synthesize easily degraded starch granules in maize seeds. The review provides a clue for developing special maize cultivars as raw material in the bioethanol industry.

On the cluster structure of amylopectin.

KEY MESSAGE: Two opposing models for the amylopectin structure are historically and comprehensively reviewed, which leads us to a better understanding of the specific fine structure of amylopectin. Amylopectin is a highly branched glucan which accounts for approximately 65-85 of starch in most plant tissues. However, its fine structure is still not fully understood due to the limitations of current methodologies. Since the 1940 s, many scientists have attempted to elucidate the distinct structure of amylopectin. One of the most accepted concepts is that amylopectin has a structural element known as "cluster", in which neighboring side chains with a degree of polymerization of ≥ 10 in the region of their non-branched segments form double helices. The double helical structures are arranged in inter- and intra-clusters and are the origin of the distinct physicochemical and crystalline properties of starch granules. Several models of the cluster structure have been proposed by starch scientists worldwide during the progress of analytical methods, whereas no direct evidence so far has been provided. Recently, Bertoft and colleagues proposed a new model designated as "the building block and backbone (BB) model". The BB model sharply contrasts with the cluster model in that the structural element for the BB model is the building block, and that long chains are separately synthesized and positioned from short chains constituting the building block. In the present paper, we conduct the historical review of the cluster concept detailing how and when the concept was established based on experimental results by many scientists. Then, differences between the two opposing concepts are explained and both models are critically discussed, particularly from the point of view of the biochemical regulation of amylopectin biosynthesis.

Changes in fine structure of amylopectin and internal structures of starch granules in developing endosperms and culms caused by starch branching enzyme mutations of japonica rice.

KEY MESSAGE: BEIIb plays a specific role in determining the structure of amylopectin in rice endosperm, whereas BEIIa plays the similar role in the culm where BEIIb is absent. Cereals have three types of starch branching enzymes (BEs), BEI, BEIIa, and BEIIb. It is widely known that BEIIb is specifically expressed in the endosperm and plays a distinct role in the structure of amylopectin because in its absence the amylopectin type changes to the amylose-extender-type (ae-type) or B-type from the wild-type or A-type and this causes the starch crystalline allomorph to the B-type from the wild-type A-type. This study aimed to clarify the role of BEIIa in the culm where BEIIb is not expressed, by using a be2a mutant in comparison with results with be2b and be1 mutants. The results showed that the amylopectin structure exhibited the B-type in the be2a culm compared with the A-type in the wild-type culm. The starch granules from the be2a culm also showed the B-type like allomorph when examined by X-ray diffraction analysis and optical sum frequency generation spectroscopy. Both amylopectin chain-length profile and starch crystalline properties were found to be the A-type at the very early stage of endosperm development at 4-6 days after pollination (DAP) even in the be2b mutant. All these results support a view that in the culm as well as in the endosperm at 4-6 DAP, BEIIa can play the role of BEIIb which has been well documented in maturing endosperm. The possible mechanism as to how BEIIa can play its role is discussed.

Protein targeting to starch 1, a functional protein of starch biosynthesis in wheat (Triticum aestivum L.).

KEY MESSAGE: TaPTST1, a wheat homolog of AtPTST1 containing CBM can interact with GBSSI and regulate starch metabolism in wheat endosperm. In cereal endosperm, native starch comprising amylose and amylopectin is synthesized by the coordinated activities of several pathway enzymes. Amylose in starch influences its physio-chemical properties resulting in several human health benefits. The Granule-Bound Starch Synthase I (GBSSI) is the most abundant starch-associated protein. GBSSI lacks dedicated Carbohydrate-binding module (CBM). Previously, Protein Targeting To Starch 1 (PTST1) was identified as a crucial protein for the localization of GBSSI to the starch granules in Arabidopsis. The function of its homologous protein in the wheat endosperm is not known. In this study, TaPTST1, an AtPTST1 homolog, containing a CBM and a coiled-coil domain was identified in wheat. Protein-coding nucleotide sequence of TaPTST1 from Indian wheat variety 'C 306' was cloned and characterized. Homology modelling and molecular docking suggested the potential interaction of TaPTST1 with glucans and GBSSI. The TaPTST1 expression was higher in wheat grain than the other tissues, suggesting a grain-specific function. In vitro binding assays demonstrated different binding affinities of TaPTST1 for native starch, amylose, and amylopectin. Furthermore, the immunoaffinity pull-down assay revealed that TaPTST1 directly interacts with GBSSI, and the interaction is mediated by a coiled-coil domain. The direct protein-protein interaction was further confirmed by bimolecular fluorescence complementation assay (BiFC) in planta. Based on our findings we postulate a functional role for TaPTST1 in starch metabolism by targeting GBSSI to starch granules in wheat endosperm.

CBM20CP, a novel functional protein of starch metabolism in green algae.

Ostreococcus tauri is a picoalga that contains a small and compact genome, which resembles that of higher plants in the multiplicity of enzymes involved in starch synthesis (ADP-glucose pyrophosphorylase, ADPGlc PPase; granule bound starch synthase, GBSS; starch synthases, SSI, SSII, SSIII; and starch branching enzyme, SBE, between others), except starch synthase IV (SSIV). Although its genome is fully sequenced, there are still many genes and proteins to which no function was assigned. Here, we identify the OT_ostta06g01880 gene that encodes CBM20CP, a plastidial protein which contains a central carbohydrate binding domain of the CBM20 family, and a coiled coil domain at the C-terminus that lacks catalytic activity. We demonstrate that CBM20CP has the ability to bind starch, amylose and amylopectin with different affinities. Furthermore, this protein interacts with OsttaSSIII-B, increasing its binding to starch granules, its catalytic efficiency and promoting granule growth. The results allow us to postulate a functional role for CBM20CP in starch metabolism in green algae. KEY MESSAGE: CBM20CP, a plastidial protein that has a modular structure but lacks catalytic activity, regulates the synthesis of starch in Ostreococcus tauri.

Suppressed expression of starch branching enzyme 1 and 2 increases resistant starch and amylose content and modifies amylopectin structure in cassava.

KEY MESSAGE: Suppression of starch branching enzymes 1 and 2 in cassava leads to increased resistant starch content through the production of high-amylose and modification of the amylopectin structure. Cassava (Manihot esculenta Crantz) is a starchy root crop used for human consumption as a staple food and industrial applications. Starch is synthesized by various isoforms of several enzymes. However, the function of starch branching enzymes (SBEs) in starch biosynthesis and mechanisms of starch regulation in cassava have not been understood well. In this study, we aimed to suppress the expression of SBEs in cassava to generate starches with a range of distinct properties, in addition to verifying the functional characteristics of the SBEs. One SBE1, two SBE2, and one SBE3 genes were classified by phylogenetic analysis and amino acid alignment. Quantitative real-time RT-PCR revealed tissue-specific expression of SBE genes in the tuberous roots and leaves of cassava. We introduced RNAi constructs containing fragments of SBE1, SBE2, or both genes into cassava by Agrobacterium-mediated transformation, and assessed enzymatic activity of SBE using tuberous roots and leaves from these transgenic plants. Simultaneous suppression of SBE1 and SBE2 rendered an extreme starch phenotype compared to suppression of SBE2 alone. Degree of polymerization of 6-13 chains in amylopectin was markedly reduced by suppression of both SBE1 and SBE2 in comparison to the SBE2 suppression; however, no change in chain-length profiles was observed in the SBE1 suppression alone. The role of SBE1 and SBE2 may have functional overlap in the storage tissue of cassava. Simultaneous suppression of SBE1 and SBE2 resulted in highly resistant starch with increased apparent amylose content compared to suppression of SBE2 alone. This study provides valuable information for understanding starch biosynthesis and suggests targets for altering starch quality.

Starch synthase II plays a crucial role in starch biosynthesis and the formation of multienzyme complexes in cassava storage roots.

Starch is a glucose polymer synthesized by green plants for energy storage and is crucial for plant growth and reproduction. The biosynthesis of starch polysaccharides is mediated by members of the large starch synthase (SS) protein superfamily. Here, we showed that in cassava storage roots, soluble starch synthase II (MeSSII) plays an important role in starch biosynthesis and the formation of protein complexes with other starch biosynthetic enzymes by directly interacting with MeSSI, MeSBEII, and MeISAII. MeSSII-RNAi cassava lines showed increased amylose content and reduced biosynthesis of the intermediate chain of amylopectin (B1 type) in their storage roots, leading to altered starch physicochemical properties. Furthermore, gel permeation chromatography analysis of starch biosynthetic enzymes between wild type and MeSSII-RNAi lines confirmed the key role of MeSSII in the organization of heteromeric starch synthetic protein complexes. The lack of MeSSII in cassava also reduced the capacity of MeSSI, MeSBEII, MeISAI, and MeISAII to bind to starch granules. These findings shed light on the key components of the starch biosynthesis machinery in root crops.

Mutation of TL1, encoding a novel C2H2 zinc finger protein, improves grains eating and cooking quality in rice.

KEY MESSAGE: The cloning and characterization of a novel C2H2 zinc finger protein that affects rice eating and cooking quality by regulating amylose content and amylopectin chain-length distribution in rice. One of the major objectives in rice breeding aims to increase simultaneously yield and grain quality especially eating and cooking quality (ECQ). Controlling amylose content (AC) and amylopectin chain-length distribution (ACLD) in rice is a major strategy for improving rice ECQ. Previous studies show that some starch synthesis-related genes (SSRGs) are required for normal AC and ACLD, but its underlying regulating network is still unclear. Here, we report the cloning and characterization of a novel C2H2 zinc finger protein TL1 (Translucent endosperm 1) that positively regulates amylose synthesis in rice grains. Loss of TL1 function reduced apparent amylose content (AAC), total starch, gel consistency, and gelatinisation temperature, whereas increased viscosity, total lipid, and ratio of amylopectin A chains with degree of polymerization (DP) 6-12 to B1 chains with DP 13-24, resulting in an enhanced grain ECQ. The improved ECQ was accompanied by altered expression patterns of several tested SSRGs in tl1 mutant grains. Furthermore, knockout of TL1 in the high-yielding rice variety JiaHua NO.1 reduced AAC without obvious side effects on major agronomic traits. These findings expand our understanding of the regulating networks of grain starch metabolism and provide new insights into how rice ECQ quality can be improved via genetic approach.

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.

CRISPR/Cas9-Mediated Mutagenesis of the Granule-Bound Starch Synthase Gene in the Potato Variety Yukon Gold to Obtain Amylose-Free Starch in Tubers.

Potato (Solanum tuberosum L.) is the third most important food crop after rice and wheat. Its tubers are a rich source of dietary carbohydrates in the form of starch, which has many industrial applications. Starch is composed of two polysaccharides, amylose and amylopectin, and their ratios determine different properties and functionalities. Potato varieties with higher amylopectin have many food processing and industrial applications. Using Agrobacterium-mediated transformation, we delivered Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) reagents to potato (variety Yukon Gold) cells to disrupt the granule-bound starch synthase (gbssI) gene with the aim of eliminating the amylose component of starch. Lugol-Iodine staining of the tubers showed a reduction or complete elimination of amylose in some of the edited events. These results were further confirmed by the perchloric acid and enzymatic methods. One event (T2-7) showed mutations in all four gbss alleles and total elimination of amylose from the tubers. Viscosity profiles of the tuber starch from six different knockout events were determined using a Rapid Visco Analyzer (RVA), and the values reflected the amylopectin/amylose ratio. Follow-up studies will focus on eliminating the CRISPR components from the events and on evaluating the potential of clones with various amylose/amylopectin ratios for food processing and other industrial applications.

Cooperative Kinetics of the Glucan Phosphatase Starch Excess4.

Glucan phosphatases are members of a functionally diverse family of dual-specificity phosphatase (DSP) enzymes. The plant glucan phosphatase Starch Excess4 (SEX4) binds and dephosphorylates glucans, contributing to processive starch degradation in the chloroplast at night. Little is known about the complex kinetics of SEX4 when acting on its complex physiologically relevant glucan substrate. Therefore, we explored the kinetics of SEX4 against both insoluble starch and soluble amylopectin glucan substrates. SEX4 displays robust activity and a unique sigmoidal kinetic response to amylopectin, characterized by a Hill coefficient of 2.77 ± 0.63, a signature feature of cooperativity. We investigated the basis for this positive kinetic cooperativity and determined that the SEX4 carbohydrate-binding module (CBM) dramatically influences the binding cooperativity and substrate transformation rates. These findings provide insights into a previously unknown but important regulatory role for SEX4 in reversible starch phosphorylation and further advances our understanding of atypical kinetic mechanisms.