Branching Enzyme Research Articles

Proteomics analysis of round and wrinkled pea (Pisum sativum L.) seeds during different development periods.

Seed development is complex, influenced by genetic and environmental factors. Understanding proteome profiles at different seed developmental stages is key to improving seed composition and quality. We used label-free quantitative proteomics to analyze round and wrinkled pea seeds at five growth stages: 4, 7, 12, 15, and days after anthesis (DAA), and at maturity. Wrinkled peas had lower starch content (30%) compared to round peas (47%-55%). Proteomic analysis identified 3659 protein groups, with 21%-24% shared across growth stages. More proteins were identified during early seed development than at maturity. Statistical analysis found 735 significantly different proteins between wrinkled and round seeds, regardless of the growth stage. The detected proteins were categorized into 31 functional classes, including metabolic enzymes, proteins involved in protein biosynthesis and homeostasis, carbohydrate metabolism, and cell division. Cell division-related proteins were more abundant in early stages, while storage proteins were more abundant later in seed development. Wrinkled seeds had lower levels of the starch-branching enzyme (SBEI), which is essential for amylopectin biosynthesis. Seed storage proteins like legumin and albumin (PA2) were more abundant in round peas, whereas vicilin was more prevalent in wrinkled peas. This study enhances our understanding of seed development in round and wrinkled peas. The study highlighted the seed growth patterns and protein profiles in round and wrinkled peas during seed development. It showed how protein accumulation changed, particularly focusing on proteins implicated in cell division, seed reserve metabolism, as well as storage proteins and protease inhibitors. These findings underscore the crucial role of these proteins in seed development. By linking the proteins identified to Cameor-based pea reference genome, our research can open avenues for deeper investigations into individual proteins, facilitate their practical application in crop improvement, and advance our knowledge of seed development.

Deciphering Biosynthesis Mechanism and Solution Properties of Cyclic Amylopectin

A novel cyclic amylopectin (CA) was synthesized from waxy corn starch (WCS) using Bacillus stearothermophilus branching enzyme (BstBE), providing insights into its biosynthesis mechanism and solution properties. During the first 4 h, BstBE partially cyclized WCS, producing 68.20% CA with a significantly reduced molecular weight (MW), from 8.98 × 10⁶ to 3.19 × 10⁴ g/mol and a lower polymer dispersity index (PDI), decreasing from 1.97 to 1.12. This resulted in a uniform CA structure with shorter chain lengths, particularly increasing DP 3–13, especially DP 7–9. Over the subsequent 4–12 h, the PDI slightly increased to 1.18 as the CA content decreased to 50.48%, with an increase in small ring structures (DP 6–12) of CA, suggesting both ring-opening and ring-downsizing due to continued enzyme catalysis. These results propose a two-stage reaction model: initial cyclization followed bybranching and secondary cyclization. CA exhibited excellent solution properties, with BE-4 and BE-12 samples demonstrating high solubility (≥65 g/100 mL), low viscosity (<0.01 Pa·s), and over 90% light transmittance after 14 days at 4 °C, highlighting its broad application potential.

Exploring the synergy of enzymes, nutrients, and gene networks in rice starch granule biogenesis.

Rice is a primary food source almost for more than 50% of the total world's population. Glycemic index (GI) is high in most of the rice varieties, limiting their consumption by diabetic and obese people. As a result, developing new rice varieties with low GI necessitates a thorough understanding of starch biogenesis gene expression and its interrelationship. A total 200 rice genotypes were analyzed for total starch content (TSC), amylopectin content (APC), and amylose content (AC). The clustering of these rice genotypes was done based on their AC. Further, these genotypes were categorized into three groups up to 10% amylose-low, 10-26% amylose-medium, and more than 26% amylose-high. Among them, six genotypes 1 from low AC (NJ-72), 2 from medium AC (UPRI-2003-18, PRR-126), and 3 from high AC (RNRM-7, Urvashi and Ananga) were selected. The genotypes selected from the medium and high AC groups were having 2% amylose variation among themselves respectively and they were further used to study the level of RS, protein content (PC), fatty acid (FA) profiles, and granule morphology along with low group sample. Resistant starch (RS) content ranged from 0.33-2.75%, and fatty acid profiling revealed high levels of palmitic, linoleic, and oleic acids. The degree of crystallinity and APC% were found to be positively correlated. Ananga, the genotype with the highest RS, displayed compact starch granules. Further, NJ-72 showing low RS and Ananga with high RS were selected for investigation of enzymatic activities of starch biosynthesis, metabolites accumulation, and expressions of 20 starch biogenesis genes in developing endosperm. Starch branching enzymes (SBE) and starch synthase (SS) activities peaked at 13 days after anthesis (DAA), while starch debranching enzymes (DBE) were most active at 18 DAA. In Ananga, TSC, AC, APC, and RS levels progressively increased from 3 to 23 DAA. Ananga showed 1.25-fold upregulation of granule-bound starch synthase I (GBSSI) at 18DAA. Higher expressions of SSI and SBEIIb were observed in NJ-72 at 13DAA. PUL2 was predominantly expressed followed by ISA1. GBSSI was positively correlated with both AC and RS while SS, SBE, and DBE were positively related to APC. This research could lead to the development of rice varieties with improved nutritional qualities, such as higher RS content, which is beneficial for human health due to its role in lowering glycemic response and promoting gut health. Additionally, the study provides insights into how the modulation of key genes and enzymes can affect starch composition, offering strategies to breed rice varieties tailored for specific dietary needs or industrial applications.

Response of Different Exogenous Phytohormones to Rice Yield Under Low-Temperature Stress at the Filling Stage

This paper aims to clarify the effects of different exogenous phytohormones on the physiological traits of rice (Oryza sativa L.) at the early stage of irrigation under low-temperature stress. In this study, two types of rice varieties with different temperature sensitivities screened out previously, namely, a cold-tolerant variety (Nan Jing 9108) and a low-temperature-sensitive variety (Hui Liang You 898), were used in pots to simulate the process of low-temperature stress in rice at the early stage of grouting (6–9 days after anthesis) with artificial low-temperature treatments. The experimental treatments were 450 mg L−1 Methyl jasmine acid (MJ), 46 mg L−1 Melatonin (MT), 69 mg L−1 Salicylate (SA), 40 mg L−1 Erythromycin (GA3), 25 mg L−1 Zeatin (Z), 145 mg L−1 Spermidine (SPD), and 5 mg L−1 Abscisic acid (ABA) sprayed on rice before low-temperature stress, while low-temperature treatment without spraying (DK) and conventional planting without spraying (CK) were added as the control. The results showed that compared with the room temperature control (CK, sprayed with deionized water), the low-temperature control (DK, low-temperature treatment, and sprayed with deionized water) all significantly reduced the rice grain yield. Different exogenous hormones sprayed before low-temperature stress could increase rice yield, among which, Z and SPD spraying treatments had a better effect on the yield of Hui Liang You 898, while different exogenous hormone treatments increased the yield of Nan Jing 9108 in an average manner. The Z and SPD treatments increased the yield of Hui Liang You 898 by 24.87% and 26.16% and that of Nan Jing 9108 by 15.87% and 17.80%, respectively. This was mainly attributed to the significant increase in thousand-grain weight and fruiting rate, while there was no significant difference in the number of spikes and number of grains. The different exogenous hormone treatments were able to delay leaf senescence, enhance the photosynthetic production capacity of plants by increasing leaf chlorophyll content, and thus increase the accumulation of photosynthetic assimilation products and population growth rate after flowering. Among them, both Z and SPD treatments resulted in a population growth rate of more than 30% from spike flushing to maturity, which led to a higher dry matter accumulation of the plant at maturity. In addition, in the dry matter distribution of the plant at maturity, the seeds occupied a higher accumulation amount and proportion compared with the respective DK; the SPD treatment resulted in the maximum distribution rate of seeds at maturity of Hui Liang You 898, with an increase of 8.27%, and the Z treatment resulted in the maximum distribution rate of seeds at maturity of Nan Jing 9108, with an increase of 7.34%. At the same time, the Z treatment significantly increased the activities of adenosine diphosphate glucose phosphorylated enzyme (AGP) and starch branching enzyme (SBE) in the grains of both varieties, which resulted in the accumulation of more starch and ultimately increased the rice grain yield. The results verified that different exogenous phytohormones could be used to regulate the insufficiency of grouting caused by low-temperature stress during the grouting and fruiting stages of rice and enriched their agronomic and physiological traits in response at the same time.

Nitrogen Level Impacts the Dynamic Changes in Nitrogen Metabolism, and Carbohydrate and Anthocyanin Biosynthesis Improves the Kernel Nutritional Quality of Purple Waxy Maize

Waxy corn is a special type of maize primarily consumed as a fresh vegetable by humans. Nitrogen (N) plays an essential role in regulating the growth progression, maturation, yield, and quality of waxy maize. A reasonable N application rate is vital for boosting the accumulation of both N and carbon (C) in the grains, thereby synergistically enhancing the grain quality. However, the impact of varying N levels on the dynamic changes in N metabolism, carbohydrate formation, and anthocyanin synthesis in purple waxy corn kernels, as well as the regulatory relationships among these processes, remains unclear. To explore the effects of varying N application rates on the N metabolism, carbohydrate formation, and anthocyanin synthesis in kernels during grain filling, a two-year field experiment was carried out using the purple waxy maize variety Jinnuo20 (JN20). This study examined the different N levels, specifically 0 (N0), 120 (N1), 240 (N2), and 360 (N3) kg N ha−1. The results of the analysis revealed that, for nearly all traits measured, the N application rate of N2 was the most suitable. Compared to the N0 treatment, the accumulation and content of anthocyanins, total nitrogen, soluble sugars, amylopectin, and C/N ratio in grains increased by an average of 35.62%, 11.49%, 12.84%, 23.74%, 13.00%, and 1.87% under N2 treatment over five filling stages within two years, respectively, while the harmful compound nitrite content only increased by an average of 30.2%. Correspondingly, the activities of related enzymes also significantly increased and were maintained under N2 treatment compared to N0 treatment. Regression and correlation analysis results revealed that the amount of anthocyanin accumulation was highly positively correlated with the activities of phenylalanine ammonia-lyase (PAL) and flavanone 3-hydroxylase (F3H), but negatively correlated with anthocyanidin synthase (ANS) and UDP-glycose: flavonoid-3-O-glycosyltransferase (UFGT) activity, nitrate reductase (NR), and glutamine synthetase (GS) showed significant positive correlations with the total nitrogen content and lysine content, and a significant negative correlation with nitrite, while soluble sugars were negatively with ADP-glucose pyrophosphorylase (AGPase) activity, and amylopectin content was positively correlated with the activities of soluble starch synthase (SSS), starch branching enzyme (SBE), and starch debranching enzyme (SDBE), respectively. Furthermore, there were positive or negative correlations among the detected traits. Hence, a reasonable N application rate improves purple waxy corn kernel nutritional quality by regulating N metabolism, as well as carbohydrate and anthocyanin biosynthesis.

Elucidation of the noncovalent interactions driving enzyme activity guides branching enzyme engineering for α-glucan modification

Branching enzymes (BEs) confer to α-glucans, the primary energy-storage reservoir in nature, a variety of features, like slow digestion. The full catalytic cycle of BEs can be divided in six steps, namely two covalent catalytic steps involving glycosylation and transglycosylation, and four noncatalytic steps involving substrate binding and transfers (SBTs). Despite the ever-growing wealth of biochemical and structural information on BEs, clear mechanistic insights into SBTs from an industrial-performance perspective are still missing. Here, we report a Rhodothermus profundi BE (RpBE) endowed with twice as much enzymatic activity as the Rhodothermus obamensis BE currently used in industry. Furthermore, we focus on the SBTs for RpBE by means of large-scale computations supported by experiment. Engineering of the crucial positions responsible for the initial substrate-binding step improves enzymatic activity significantly, while offering a possibility to customize product types. In addition, we show that the high-efficiency substrate-transfer steps preceding glycosylation and transglycosylation are the main reason for the remarkable enzymatic activity of RpBE, suggestive of engineering directions for the BE family.

Correlation analysis of starch molecular structure and film properties via rearrangements of glycosidic linkages by 1,4-α-glucan branching enzyme

The functional characteristics of starch films are significantly influenced by the amylose content and the distribution of the amylopectin chain length. This work used 1,4-α-glucan branching enzyme to molecularly reconstruct corn, pea, and cassava starch in order to examine the association. Films made of both natural and enzyme-modified starch were produced using the casting method. The study investigated the variations in starch films properties and explored the relationship between starch molecular structure and film qualities by correlation analysis. The results showed a significant positive connection (r = 0.954) between the tensile strength and amylose content, as well as a positive correlation (r = 0.939) between the A chains and the elongation at break. The average chain length (r = 0.932) and amylose content (r = 0.902) showed a positive correlation with the degradation temperature, whereas the amylose content (r = −0.946) showed an adverse correlation with the transparency. The B3 chain (r = 0.851) and the average chain length (r = 0.839) both exhibited a positive connection with its contact angle. As a result, our study thoroughly assesses how starch structure affects the characteristics of starch films and offers a fundamental modification pathway for the development of new application areas.

The transcription factor CAMTA2 interacts with the histone acetyltransferase GCN5 and regulates grain weight in wheat.

Grain weight and size are major traits targeted in breeding to improve wheat (Triticum aestivum L.) yield. Here, we find that the histone acetyltransferase GENERAL CONTROL NONDEREPRESSIBLE 5 (GCN5) physically interacts with the calmodulin-binding transcription factor CAMTA2 and regulates wheat grain size and weight. gcn5 mutant grains were smaller and contained less starch. GCN5 promoted the expression of the starch biosynthesis genes SUCROSE SYNTHASE 2 (Sus2) and STARCH-BRANCHING ENZYME Ic (SBEIc) by regulating H3K9ac and H3K14ac levels in their promoters. Moreover, immunoprecipitation followed by mass spectrometry (IP-MS) revealed that CAMTA2 physically interacts with GCN5. The CAMTA2-GCN5 complex activated Sus2 and SBEIc by directly binding to their promoters and depositing H3K9ac and H3K14ac marks during wheat endosperm development. camta2 knockout mutants exhibited similar phenotypes to gcn5 mutants, including smaller grains that contained less starch. In gcn5 mutants, transcripts of high molecular weight (HMW) Glutenin (Glu) genes were downregulated, leading to reduced HMW glutenin protein levels, gluten content, and sodium dodecyl sulfate (SDS) sedimentation volume. However, the association of GCN5 with Glu genes was independent of CAMTA2, since GCN5 enrichment on Glu promoters was unchanged in camta2 knockouts. Finally, we identified a CAMTA2-AH3 elite allele that corresponded with enhanced grain size and weight, serving as a candidate gene for breeding wheat varieties with improved grain weight.

Synergistic action of novel maltohexaose-forming amylase and branching enzyme improves the enzymatic conversion of starch to specific maltooligosaccharide

As attractive functional ingredients, maltooligosaccharides (MOS) are typically prepared by controlled enzymatic hydrolysis of starch. However, the random attack mode of amylase often leads to discrete product distribution, thereby reducing yields and purities. In this study, a novel glycoside hydrolase family 13 amylase AmyEs from marine myxobacteria Enhygromyxa salina was identified efficient maltohexaose (G6)-forming ability (40 %, w/w). By deciphering external chain length, we found that the high density of α-1,6-branching points benefits the G6 formation of AmyEs with high purity (71–82 %), indicating the substrate selectivity of AmyEs toward high-branched starch. Based on this, asynchronous conversion strategy was designed to enhance specific MOS yield from corn starch by exploiting branching enzymes and AmyEs, and the purity and yield of G6 respectively increased by 9.5 % and 5 % compared to single AmyEs treatment. Our results demonstrate that combinatorial catalysis of MOS-forming amylases and branching enzymes provides a favorable industrial preparation of specific MOS.

PSVIII-3 The functional properties and digestibility of round and wrinkled pea flour in swine diets

Abstract Wrinkled and round pea flour display physical and compositional differences. Wrinkled peas have reduced activity of the starch branching enzyme, which decreases amylopectin production. As a result, wrinkled peas have an increased proportion of amylose, relative to round peas. The rate and extent of starch digestion are influenced by the ratio of amylose to amylopectin in starch. Extrusion results in swelling of the starch, altering the crystalline structure. The objective of this study was to determine the role of the amylose to amylopectin ratio and extrusion on starch digestibility and microbial characteristics in growing pigs. Ileal-cannulated barrows (n = 8) with an initial body weight (BW) of 40 ± 1 kg were randomly assigned to 1 of 4 dietary treatments (n = 8 pigs/treatment) in a replicated Latin square design with four blocks. Dietary treatments were round pea flour (RPF), extruded round pea flour (RPF-E), wrinkled pea flour (WPF), and extruded wrinkled pea flour (WPF-E). The starch content decreased in the native WPF (36.7%) and WPF-E (34.7%) compared with RPF (50.1%) and RPF-E (49.1%). However, the amylose content in native WPF (77.1%) and WPF-E (70.7%) was greater compared with RPF (40.1%) and RPF-E (39.8%). Ileal digestibility of starch differed between pea flours (P < 0.05), greatest for RPF (92.8%) and RPF-E (92.2%) and least for WPF (80.2%) and WPF-E (76.7%). Total tract digestibility was least for RPF (98.7%) compared with other treatments (P < 0.05). Total tract digestibility of starch was greatest with RPF-E compared with RPF (99.5% vs 98.7%, P < 0.01) but was not different from WPF (99.25%) or WPF-E (99.51%). Shannon index, an indicator of microbial diversity, decreased in ileal digesta of WPF-E-fed pigs compared with RPF-E (P < 0.05) but similar among treatments in the feces. In summary, compared with WPF or WPF-E, RPF and RPF-E had reduced amylose content and greater starch digestibility determined at the end of the ileum; however, total tract digestibility of starch was similar among all treatments. Furthermore, RPF-E compared with RPF, WPF, and WPF-E increased microbial diversity in the ileal digesta, but no difference among treatments in the feces.

Preparation of Polyclonal Antibodies to Barley Granule-Bound Amylopectin Synthase Ia and Their Application in the Characterization of Interacting Proteins

The production of amylose is facilitated by granule-bound starch synthase (GBSS). Despite its importance, the specific protein interactions involving barley grain-bound starch synthase Ia (HvGBSSIa) remain poorly understood. To elucidate this, we engineered a pET-32a-HvGBSSIa prokaryotic expression vector for specific expression in E. coli Rosetta cells. A rabbit anti-HvGBSSIa polyclonal antibody was generated and employed to enrich HvGBSSIa-binding proteins from barley grains through immunoprecipitation. The isolated complexes were then resolved through SDS-PAGE, and the constituent proteins were identified using mass spectrometry coupled with database searches. Our results confirmed the successful preparation of a highly specific polyclonal antibody against HvGBSSI. Furthermore, differential expression of HvGBSSIa was assessed across various barley tissues and developmental stages of the grain, revealing peak expression at 25 days post-flowering. Proteins interacting with HvGBSSIa, including sucrose synthase and starch branching enzyme, were identified through co-immunoprecipitation. This study lays the groundwork for further detailed analyses of the HvGBSSIa protein complex in barley.

Mutations in starch BRANCHING ENZYME 2a suppress the traits caused by the loss of ISOAMYLASE1 in barley

Key messageThe hvbe2a mutations restore the starch-deficient phenotype caused by the hvisa1 and hvflo6 mutations in barley endosperm.The genetic interactions among starch biosynthesis genes can be exploited to alter starch properties, but they remain poorly understood due to the various combinations of mutations to be tested. Here, we isolated two novel barley mutants defective in starch BRANCHING ENZYME 2a (hvbe2a-1 and hvbe2a-2) based on the starch granule (SG) morphology. Both hvbe2a mutants showed elongated SGs in the endosperm and increased resistant starch content. hvbe2a-1 had a base change in HvBE2a gene, substituting the amino acid essential for its enzyme activity, while hvbe2a-2 is completely missing HvBE2a due to a chromosomal deletion. Further genetic crosses with barley isoamylase1 mutants (hvisa1) revealed that both hvbe2a mutations could suppress defects in endosperm caused by hvisa1, such as reduction in starch, increase in phytoglycogen, and changes in the glucan chain length distribution. Remarkably, hvbe2a mutations also transformed the endosperm SG morphology from the compound SG caused by hvisa1 to bimodal simple SGs, resembling that of wild-type barley. The suppressive impact was in competition with floury endosperm 6 mutation (hvflo6), which could enhance the phenotype of hvisa1 in the endosperm. In contrast, the compound SG formation induced by the hvflo6 hvisa1 mutation in pollen was not suppressed by hvbe2a mutations. Our findings provide new insights into genetic interactions in the starch biosynthetic pathway, demonstrating how specific genetic alterations can influence starch properties and SG morphology, with potential applications in cereal breeding for desired starch properties.

Multiomics of a rice population identifies genes and genomic regions that bestow low glycemic index and high protein content

To counter the rising incidence of diabetes and to meet the daily protein needs, we created low glycemic index (GI) rice varieties with protein content (PC) surpassing 14%. In the development of recombinant inbred lines using Samba Mahsuri and IR36 amylose extender (IR36ae) as parental lines, we identified quantitative trait loci and genes associated with low GI, high amylose content (AC), and high PC. By integrating genetic techniques with classification models, this comprehensive approach identified candidate genes on chromosome 2 (qGI2.1/qAC2.1 spanning the region from 18.62 Mb to 19.95 Mb), exerting influence on low GI and high amylose. Notably, the phenotypic variant with high value was associated with the recessive allele of the starch branching enzyme 2b (sbeIIb). The genome-edited sbeIIb line confirmed low GI phenotype in milled rice grains. Further, combinations of alleles created by the highly significant SNPs from the targeted associations and epistatically interacting genes showed ultralow GI phenotypes with high amylose and high protein. Metabolomics analysis of rice with varying AC, PC, and GI revealed that the superior lines of high AC and PC, and low GI were preferentially enriched in glycolytic and amino acid metabolisms, whereas the inferior lines of low AC and PC and high GI were enriched with fatty acid metabolism. The high amylose high protein recombinant inbred line (HAHP_101) was enriched in essential amino acids like lysine. Such lines may be highly relevant for food product development to address diabetes and malnutrition.

NnSBE1 encodes a starch branching enzyme involved in starch biosynthesis in lotus seeds

Lotus seed starch holds vast potential for utilization across various industries, with its content and structure directly influencing the commercial value of lotus seeds. However, there has been limited information available on the molecular mechanisms underlying lotus seed starch biosynthesis. In this study, three starch branching enzyme homologs were identified in the lotus genome, designated as NnSBE1 to NnSBE3, which possess conserved CBM_48 and α_Aamy domains. Among them, NnSBE1 exhibited predominant expression, with abundant transcript levels observed in lotus seeds and flower-related organs. Expression of NnSBE1 remained consistently up-regulated in lotus cotyledons from 6 to 21 days after pollination. Additionally, a C2H2-type finger protein encoding gene, NnLOL1, co-expressed with NnSBE1 in lotus cotyledons. As a seed-predominantly expressed transcription factor, NnLOL1 was confirmed to activate NnSBE1 expression. Transient overexpression of NnSBE1 in lotus cotyledons resulted in a significant increase in both amylopectin and total starch content compared to the control. Furthermore, multiple variation sites within the NnSBE1 gene gave rise to diverse haplotypes between seed-lotus and other lotus varieties. These findings contribute to our understanding of the regulation mechanisms involved in lotus seed starch biosynthesis, offering valuable theoretical insights for the genetic improvement of lotus seed starch by molecular breeding strategies.

Effect of branch length of cluster dextrin on the textural and rheological properties of κ-carrageenan emulsion gels

In this paper, a range of novel cluster dextrins (CDs) with high molecular weight (∼105 g/mol) and narrow molecular weight distribution were fabricated by α-amylase and branching enzyme (BE, EC 2.4.1.18). The influence of CDs on the physicochemical properties of κ-carrageenan (KC) emulsion gels was investigated. The average particle size of the composite emulsion gels containing CD was decreased by 7.85–12.66 μm, and the gel network was more uniform and denser, owing to stronger non-covalent interactions in the composite sample system. Textural results demonstrated that the composite emulsion gels exhibited higher hardness and springiness, but lower cohesiveness. CDs hindered the formation and aggregation of KC double helices, as evidenced by reducing the intensity of the sulfate group peak at 1223 cm−1. Furthermore, CDs increased the apparent viscosity, storage modulus (G'), and melting temperature (Tm) of emulsion gels, based on rheological results. CDs with longer branch lengths were more prone to entangle with KC chains to optimize the gel structure, exhibiting a more significant effect on the physicochemical properties of emulsion gels. The present study provided a new insight for constructing a new energy delivery system.

Neuro-Ophthalmic Manifestations of Adult Polyglucosan Body Disease.

Adult polyglucosan body disease (APBD) is caused by a deficiency in glycogen branching enzyme that leads to polyglucosan accumulation in multiple organs. It has a progressive clinical course with prominent neurologic manifestations. We aim to describe the neuro-ophthalmic manifestations of APBD. This is a case series of 3 individuals with genetically proven APBD. Written informed consent was provided by the brothers. We also performed a literature review on the current state of knowledge on APBD through PubMed. Brother 1 developed gait imbalance and length-dependent polyneuropathy in his 40s followed by progressive urinary symptoms in his 50s. He reported diplopia and blurry vision in his 60s. Neuro-ophthalmic assessment revealed bilateral optic neuropathy, convergence insufficiency, and a right fourth nerve palsy. Genetic testing showed a homozygous pathogenic variant in GBE1 c.986A>C p.Tyr329Ser. Brother 2 developed progressive urinary symptoms in his 40s that were followed by cognitive deficits, length-dependent polyneuropathy, and lower extremity weakness in his 50s and 60s. He reported blurred vision, and neuro-ophthalmic evaluation revealed bilateral optic neuropathy. Genetic testing revealed the same variant as Brother 1, GBE1 c.986A>C p.Tyr329Ser. Brother 3 developed progressive urinary urgency and lower extremity weakness in his 50s followed by a length-dependent polyneuropathy in his 60s. He reported diplopia and blurry vision in his 70s. Neuro-ophthalmic assessment revealed bilateral optic neuropathy and convergence insufficiency. Genetic testing revealed the same variant as Brothers 1 and 2, GBE1 c.986A>C p.Tyr329Ser. There is an array of afferent and efferent neuro-ophthalmic manifestations in APBD. Neuro-ophthalmic evaluation is crucial in evaluating and treating patients with APBD, particularly in those with visual dysfunction.

Protein kinase MeSnRK2.3 positively regulates starch biosynthesis by interacting with the transcription factor MebHLH68 in cassava.

Starch biosynthesis involves numerous enzymes and is a crucial metabolic activity in plant storage organs. Sucrose non-fermenting related protein kinase 2 (SnRK2) is an abscisic acid (ABA)-dependent kinase and a significant regulatory enzyme in the ABA signaling pathway. However, whether SnRK2 kinases regulate starch biosynthesis is unclear. In this study, we identified that MeSnRK2.3, an ABA-dependent kinase, was highly expressed in the storage roots of cassava and was induced by ABA. Overexpression of MeSnRK2.3 in cassava significantly increased the starch content in the storage roots and promoted plant growth. MeSnRK2.3 was further found to interact with the cassava basic helix-loop-helix 68 (MebHLH68) transcription factor in vivo and in vitro. MebHLH68 directly bound to the promoters of sucrose synthase 1 (MeSUS1), granule-bound starch synthase I a (MeGBSSIa), and starch-branching enzyme 2.4 (MeSBE2.4), thereby upregulating their transcriptional activities. Additionally, MebHLH68 negatively regulated the transcriptional activity of sucrose phosphate synthase B (MeSPSB). Moreover, phosphorylated MebHLH68 by MeSnRK2.3 up-regulated the transcription activity of MeSBE2.4. These findings demonstrated that the MeSnRK2.3-MebHLH68 module connects the ABA signaling pathway and starch biosynthesis in cassava, thereby providing direct evidence of ABA-mediated participation in the sucrose metabolism and starch biosynthesis pathway.

Biochar Improves Yield by Reducing Saline-Alkaline Stress, Enhancing Filling Rate of Rice in Soda Saline-Alkaline Paddy Fields.

Soda saline-alkaline stress significantly impedes the rice grain filling process and ultimately impacts rice yield. Biochar has been shown to mitigate the negative impacts of saline-alkaline stress on plants. However, the exact mechanism by which biochar influences the rice grain-filling rate in soda saline-alkaline soil is still not fully understood. A two-year field experiment was conducted with two nitrogen fertilizer levels (0 and 225 kg ha-1) and five biochar application rates [0% (B0), 0.5% (B1), 1.5% (B2), 3.0% (B3), and 4.5% (B4) biochar, w/w]. The results demonstrated that biochar had a significant impact on reducing the Na+ concentration and Na+/K+ ratio in rice grown in soda saline-alkaline lands, while also improving its stress physiological conditions. B1, B2, B3, and B4 showed a notable increase in the average grain-filling rate by 5.76%, 6.59%, 9.80%, and 10.79%, respectively, compared to B0; the time to reach the maximum grain-filling rate and the maximum grain weight saw increases ranging from 6.02% to 12.47% and from 7.85% to 14.68%, respectively. Meanwhile, biochar, particularly when used in conjunction with nitrogen fertilizer, notably enhanced the activities of sucrose synthase (SuSase), ADPG pyrophosphorylase (AGPase), starch synthase (StSase), and starch branching enzyme (SBE) of rice grains in soda saline-alkaline lands. Furthermore, rice yield increased by 11.95-42.74% in the B1, B2, B3, and B4 treatments compared to the B0 treatment. These findings showed that biochar improves yield by regulating ionic balance, physiological indicators, starch synthesis key enzyme activities, and the grain-filling rate in soda saline-alkaline paddy fields.

The impact of Cas9-mediated mutagenesis of genes encoding potato starch-branching enzymes on starch structural properties and in vitro digestibility

The digestibility of starch is affected by amylose content, and increasing amylopectin chain length which can be manipulated by alterations to genes encoding starch-branching enzymes (SBEs). We investigated the impact of Cas9-mediated mutagenesis of SBEs in potato on starch structural properties and digestibility.Four potato starches with edited SBE genes were tested. One lacked SBE1 and SBE2, two lacked SBE2 and had reduced SBE1, and one had reduced SBE2 only. Starch structure and thermal properties were characterised by DSC and XRD. The impact of different thermal treatments on digestibility was studied using an in vitro digestion protocol.All native potato starches were resistant to digestion, and all gelatinised starches were highly digestible. SBE modified starches had higher gelatinisation temperatures than wild type potatoes and retrograded more rapidly. Gelatinisation and 18 h of retrogradation, increased gelatinisation enthalpy, but this did not translate to differences in digestion. Following 7 days of retrogradation, starch from three modified SBE starch lines was less digestible than starch from wild-type potatoes, likely due to the recrystallisation of the long amylopectin chains. Our results indicate that reductions in SBE in potato may be beneficial to health by increasing the amount of fibre reaching the colon after retrogradation.

Starch molecular structure and diabetes

Starch is a primary source of food energy for human beings. Its chain-length distribution (CLD) is a major structural feature influencing physiologically-important properties, such as digestibility and palatability, of starch-containing foods. Diabetes, which is of epidemic proportions in many countries, is related to the rate of starch digestion in foods. Isoforms of three biosynthesis enzymes, starch synthase, starch branching enzymes and debranching enzymes, control the CLDs of starch, which can be measured by methods such as size-exclusion chromatography and fluorophore-assisted carbohydrate electrophoresis. Fitting observed CLDs to biosynthesis-based models based on the ratios of the activities of those isoforms yields biosynthesis-related parameters describing CLD features. This review examines CLD measurement, fitting CLDs to models, relations between CLDs, the occurrence and management of diabetes, and how plant breeders can develop varieties to optimize digestibility and palatability together, to develop starch-based foods with both a lower risk of diabetes and acceptable taste.