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.

Understanding starch biosynthesis in potatoes for metabolic engineering to improve starch quality: A detailed review.

Potato tubers accumulate substantial quantities of starch, which serves as their primary energy reserve. As the predominant component of potato tubers, starch strongly influences tuber yield, processing quality, and nutritional attributes. Potato starch is distinguished from other food starches by its unique granule morphology and compositional attributes. It possesses large, oval granules with amylose content ranging from 20 to 33 % and high phosphorus levels, which collectively determine the unique physicochemical characteristics. These physicochemical properties direct the utility of potato starch across diverse food and industrial applications. This review synthesizes current knowledge on the molecular factors controlling potato starch biosynthesis and structure-function relationships. Key topics covered are starch granule morphology, the roles and regulation of major biosynthetic enzymes, transcriptional and hormonal control, genetic engineering strategies, and opportunities to tailor starch functionality. Elucidating the contributions of different enzymes in starch biosynthesis has enabled targeted modification of potato starch composition and properties. However, realizing the full potential of this knowledge faces challenges in optimizing starch quality without compromising plant vigor and yield. Overall, integrating multi-omics datasets with advanced genetic and metabolic engineering tools can facilitate the development of elite cultivars with enhanced starch yield and tailored functionalities.

QTL Mapping and Candidate Gene Analysis for Starch-Related Traits in Tartary Buckwheat (Fagopyrum tataricum (L.) Gaertn).

Starch is the main component that determines the yield and quality of Tartary buckwheat. As a quantitative trait, using quantitative trait locus (QTL) mapping to excavate genes associated with starch-related traits is crucial for understanding the genetic mechanisms involved in starch synthesis and molecular breeding of Tartary buckwheat varieties with high-quality starch. Employing a recombinant inbred line population as research material, this study used QTL mapping to investigate the amylose, amylopectin, and total starch contents across four distinct environments. The results identified a total of 20 QTLs spanning six chromosomes, which explained 4.07% to 14.41% of the phenotypic variation. One major QTL cluster containing three stable QTLs governing both amylose and amylopectin content, qClu-4-1, was identified and located in the physical interval of 39.85-43.34 Mbp on chromosome Ft4. Within this cluster, we predicted 239 candidate genes and analyzed their SNP/InDel mutations, expression patterns, and enriched KEGG pathways. Ultimately, five key candidate genes, namely FtPinG0004897100.01, FtPinG0002636200.01, FtPinG0009329200.01, FtPinG0007371600.01, and FtPinG0005109900.01, were highlighted, which are potentially involved in starch synthesis and regulation, paving the way for further investigative studies. This study, for the first time, utilized QTL mapping to detect major QTLs controlling amylose, amylopectin, and total starch contents in Tartary buckwheat. The QTLs and candidate genes would provide valuable insights into the genetic mechanisms underlying starch synthesis and improving starch-related traits of Tartary buckwheat.

Impact of mutations in starch synthesis genes on morphological, compositional, molecular structure, and functional properties of potato starch.

Morphology, composition and molecular structure of starch directly affect the functional properties. This study investigated the morphological, compositional, and molecular structure properties of starch from starch branching enzyme gene (SBE) and granule-bound starch synthase gene (GBSS) mutated potato, and their associations with thermal, pasting, and film-making properties. SBE mutations were induced in native variety Desiree while GBSS mutations were herestacked to a selected SBE mutated parental line. Mutations in SBE resulted in smaller starch granules and higher amylose content, while GBSS mutations in the SBE background reduced amylose content. Mutations in SBE, particularly with GBSS mutations, significantly increased total phosphorus content. 31P NMR spectroscopy revealed higher proportions of C6-bound phosphate than of C3-bound phosphate in all studied lines. Amylopectin unit chain and internal chain distributions showed higher proportions of long chains in mutated lines compared with Desiree. These amylopectin long-chains were positively correlated with gelatinizationand, pasting temperatures, and temperature at peak viscosity. Short amylopectin chains showed positive correlations with breakdown viscosity, but negative correlations with the crystal melting temperature of retrograded starch. Total phosphorus content was positively correlated with the crystal melting temperature of retrograded starch. Starch from different lines was used to produce a series of potato starch films that differed in morphology and functional properties. A negative correlation was observed between Young's modulus of films and the long amylopectin-chain fraction. Thermal gravimetric analysis revealed highest thermal stability of Desiree starch films, followed by films from SBE-mutated high-amylose lines. Oxygen transmission rate and oxygen permeability analyses showed that films made with starch from selected GBSS and SBEs mutated line maintained comparable oxygen barrier properties to Desiree film. These insights on the impact of genetic mutations on starch properties indicate potential applications of in-planta starch modification for specific end-uses including packaging.

Investigation of the link between first-order kinetic models of the in vitro digestion of native starches and the accompanying changes in their crystallinity and structure.

Starch is the main source of dietary energy for humans. In order to understand the mechanisms governing native starch in vitro digestion, digestion data for six starches [wheat, maize, (waxy) maize, rice, potato and pea] of different botanical sources were fitted with the most common first-order kinetic models, i.e. the single, sequential, parallel and combined models. Parallel and combined models provided the most accurate fits and showed that all starches studied except potato starch followed a biphasic in vitro digestion pattern. The biological relevance of the kinetic parameters was explored by determining changes in crystallinity and molecular structure of the undigested starch residues during in vitro digestion. While the crystallinity of the undigested potato starch residues did not change substantially, a respectively small and large decrease in their amylose content and chain length during in vitro digestion was observed, indicating that amylose was digested slightly preferentially over amylopectin in native starch. However, the molecular structure of the starch residues changed too slowly and/or only to an insufficient extent to relate it to the kinetic parameters of the digested fractions predicted by the models. Such parameters thus need to be interpreted with caution, as their biological relevance still needs to be proven.

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.

Modulating rice digestibility: Mechanisms of ultrasound, oleic acid and moist-heat treatments influence structural, physicochemical and digestive properties.

The research compared the combined effect of ultrasound (160 W, 2 min), oleic acid (15%, 11 h), and moist-heat treatment (HMT, 25% moisture content, 110 °C, 2 h) with their individual treatment on rice grains. The results showed that ultrasound treatment created pores and cracks in the rice grains, facilitating an easier penetration for oleic acid to develop amylose-oleic acid complex during HMT. Compared to native raw rice (NR), both single and combined treatments significantly altered the morphology, reduced swelling power and solubility, enhanced hydrophilicity, thus changing the moisture distribution, thermal and pasting characteristics. Notably, the combined treatment of three techniques significantly increased the relative crystallinity, accompanied by the highest digestive resistance, and the content of resistant starch was increased from 20.53% in NR to 31.75%, much higher than the other treatments. These findings provide potential for the manufacturers to rationally and flexibly employ this low digestible rice in health food products.

Insights into the catalytic properties of 4,3-α-glucanotransferase to guide the biofabrication of α-glucans with low digestibility.

The effect of the starch chain structure on 4,3-α-glucanotransferase's (4,3-α-GTase) catalytic properties was investigated to modulate the digestibility of starch. Three starches with diverse amylose contents were used, and the enzymatic kinetic reaction of 4,3-α-GTase was fitted using the Michaelis-Menten equation. The results revealed that the linear substrate was more suitable for modification by 4,3-α-GTase. Linear starch chains were then selected with various degrees of polymerization (DP) as substrates of 4,3-α-GTase modification. Additionally, the structures and in vitro digestion of 4,3-α-GTase derived α-glucans were studied. The results showed that enzyme catalysis increased the amount of α-1,3 glycosidic linkages in products (highest 33.5%), the digestibility of 4,3-α-GTase derived α-glucans conformed to a first-order two-phase equation, and the equilibrium digestibility was controlled between 43.2-72.1%. It was observed that the structure of α-glucans could be managed to attain low digestibilities (43.2%) by selecting maltodextrin with DE 2 as the substrate. These findings offer valuable insights into the fabrication of α-glucans and their potential applications in various fields.

Insights into starch synthesis and amino acid composition of common buckwheat in response to phosphate fertilizer management strategies.

To investigate the response and the regulatory mechanism of common buckwheat starch, amylose, and amylopectin biosynthesis to P management strategies, field experiments were conducted in 2021 and 2022 using three phosphorus (P) levels. Results revealed that the application of 75 kg hm-2 phosphate fertilizer significantly enhanced amylopectin and total starch content in common buckwheat, leading to improved grain weight and starch yield, and decreased starch granule size. The number of upregulated differentially expressed proteins induced by phosphate fertilizer increased with the application rate, with 56 proteins identified as shared differential proteins between different P levels, primarily associated with carbohydrate and amino acid metabolism. Phosphate fertilizer inhibited amylose synthesis by downregulating granule-bound starch synthase protein expression and promoted amylopectin accumulation by upregulating 1,4-alpha-glucan branching enzyme and starch synthase proteins expression. Additionally, Phosphate fertilizer primarily promoted the accumulation of hydrophobic and essential amino acids. These findings elucidate the mechanism of P-induced starch accumulation and offer insights into phosphate fertilizer management and high-quality cultivation of common buckwheat.

Improving functional and nutritional profiles of barley flours with diverse starch types through pearling.

A comparative analysis of chemical, functional, and digestive parameters was conducted on five new barley genotypes designed for food purposes, differing in starch type, ß-glucans, and arabinoxylan content. Both whole and pearled grain flours were examined. Amylose exhibited positive correlations with least gelation capacity (r = 0.60), gelation temperature (r = 0.90), and resistant starch (r = 0.80). Waxy varieties showed greater water-holding capacity, viscosity, and rapid digestibility compared to normal and high-amylose varieties. Pearling (10%) decreased arabinoxylans by 48% and proteins by 7%, while increasing ß-glucans by 8% and starch by 13%. Additionally, pearling improved viscosity and hydration parameters across varieties. This allowed normal and high-amylose genotypes to enhance their functional properties and nutritional value through increased ß-glucan and resistant starch content. This exploration advances the understanding of barley's functional attributes for food industry and underscores the potential of pearling to augment consumer nutritional value and health-promoting properties.

Transcription factor OsbZIP10 modulates rice grain quality by regulating OsGIF1.

Understanding and optimizing the process of grain filling helps the quest to maximize rice (Oryza sativa L.) seed yield and quality, yet the intricate mechanisms at play remain fragmented. Transcription factors (TFs) are major players in the gene networks underlying the grain filling process. Here, we employed grain incomplete filling (OsGIF1)/cell wall invertase 2, a key gene involved in grain filling, to explore its upstream TFs and identified a bZIP family TF, OsbZIP10, to be a transcriptional activator of OsGIF1. Rice grains of the knockouts of OsbZIP10 showed increased white-core rates but lower amylose content (AC), leading to better eating and cooking qualities in all genetic backgrounds investigated, though the impact of mutations in OsbZIP10 on grain weight depended on genetic background. Multi-omics analyses suggested that, in addition to OsGIF1, multiple genes involved in different biological processes contributing to grain filling were targeted by OsbZIP10, including OsAGPS1, a gene encoding the ADP-Glc pyrophosphorylase (AGPase) small subunit, and genes contributing to homeostasis of reactive oxygen species. Distinct genetic make-up was observed in OsbZIP10 between japonica and indica rice varieties, with the majority varieties of each subspecies belonging to two different haplotypes that were closely associated with AC. Overexpressing the haplotype linked to high-AC in the low-AC genetic background increased AC. Overall, this study sheds crucial light on the significance of the OsbZIP10-OsGIF1 module in the determination of rice grain quality, offering a potential avenue for genetic engineering of rice to produce seeds with tailored attributes.

Creation of rice doubled haploids with low amylose content using in vitro anther culture.

In vitro androgenesis is a unique model for producing homozygous doubled haploid plants. The use of haploid biotechnology accelerates to obtain of doubled haploid plants, which is very important in rice breeding. The purpose of this work is to improve the production of doubled haploids in rice anther culture in vitro and selection of doubled haploid plants with valuable traits. The study the influence of nutrient media on the production of calli and plant regeneration processes in anther culture of 35 rice genotypes was revealed a significant influence of nutrient media on callus production. It was shown that the addition to culture medium phytohormones ratio with high level of cytokinin (5.0 mg/L BAP) and a low level of auxin (0.5 mg/L NAA), supplemented with amino acid composition promotes high production of green regenerated plants (68.75%) compared to albino plants (31.25%). As a result, doubled haploid lines of the glutinous variety Violetta were selected, which characterized by a low amylose content variation (from 1.86 to 2.80%). These doubled haploids are superior to the original variety in some yield traits and represent valuable breeding material.

Combining genome-wide association study and linkage mapping in the genetic dissection of amylose content in maize (Zea mays L.).

KEY MESSAGE: Integrated linkage and association analysis revealed genetic basis across multiple environments. The genes Zm00001d003102 and Zm00001d015905 were further verified to influence amylose content using gene-based association study. Maize kernel amylose is an important source of human food and industrial raw material. However, the genetic basis underlying maize amylose content is still obscure. Herein, we used an intermated B73 × Mo17 (IBM) Syn10 doubled haploid population composed of 222 lines and a germplasm set including 305 inbred lines to uncover the genetic control for amylose content under four environments. Linkage mapping detected 16 unique QTL, among which four were individually repeatedly identified across multiple environments. Genome-wide association study revealed 17 significant (P = 2.24E-06) single-nucleotide polymorphisms, of which two (SYN19568 and PZE-105090500) were located in the intervals of the mapped QTL (qAC2 and qAC5-3), respectively. According to the two population co-localized loci, 20 genes were confirmed as the candidate genes for amylose content. Gene-based association analysis indicated that the variants in Zm00001d003102 (Beta-16-galactosyltransferase GALT29A) and Zm00001d015905 (Sugar transporter 4a) affected amylose content across multi-environment. Tissue expression analysis showed that the two genes were specifically highly expressed in the ear and stem, respectively, suggesting that they might participate in sugar transport from source to sink organs. Our study provides valuable genetic information for breeding maize varieties with high amylose.

Oleic acid treatment of rice grains reduces the starch digestibility: Formation, binding state and fine structure of starch-lipid complexes.

Rice contains abundant starch and contributes to a rapid rise in postprandial blood glucose levels. Hence, it is crucial to directly modify rice grains for resistant starch (RS) content elevation while preserving their morphology. In this study, rice grains were treated with 6%-18% concentrations of oleic acid (OA) and 8-20 h of soaking time to promote the formation of starch-lipid complexes, thereby reducing rice digestibility. In OA-treated rice, the OA molecules exist in three binding states. OA-treated rice exhibited a significantly higher complexation index and OA content than natural rice. RS content increased from 20.50% to 32.46%. X-ray diffraction and NMR spectroscopy revealed the development of amylose-OA complexes within the rice grains and a V-crystalline structure of up to 3.62%. Raman spectroscopy and thermogravimetric analysis showed enhanced molecular ordering and structural stability of rice starch. Overall, OA treatment effectively promotes RS formation within rice grains, consequently reducing rice digestibility.

Starch chain-length distributions affect the processing and digestion characteristics of proso millet starch.

Starch chain-length distributions play a key role in regulating the processing and digestion characteristics of proso millet starch. Waxy proso millet starch has higher endothermic enthalpy (13.06-16.73 J/g) owing to its higher relative crystallinity (27.83%-32.04%), while nonwaxy proso millet starch has lower peak viscosity (1.0630-1.1930 Pa∙s) and stronger viscoelasticity owing to its higher amylose content (21.72%-24.34%). Non-waxy proso millet starch exhibited two different digestion phases and its resistant starch content (18.37%-20.80%) was higher than waxy proso millet starch. Correlation analysis showed proso millet starch with longer amylopectin B1 chains and more amylopectin B2 chains exhibited excellent thermal ability and retrograde resistance, whereas proso millet starch with shorter and more amylose medium/long-chains not only reduced the digestion rate and increased the resistant starch content but also exhibited stronger viscoelasticity and excellent retrogradation properties. These results could provide more insights into efficient utilization of proso millet starch.

Effects of Genotype, Nitrogen, and Sulfur Complex Fertilization on the Nutritional and Technological Characteristics of Buckwheat Flour.

The present study investigated the effect of nitrogen fertilization (NF) at the levels of 0, 45, and 90 kg·ha-1 combined with selected sulfur complex fertilization (SCF) levels of 0 and 45 kg·ha-1 on the nutritional and technological characteristics of buckwheat flour from five varieties. The results showed that the genotype was a critical factor affecting the chemical composition and physicochemical properties of buckwheat flour. NF significantly increased protein, total starch, and amylose content as well as mineral composition but decreased particle size, color value, and water hydration properties. However, SCF enhanced the ash content and decreased the protein content but had no significant effect on the pasting temperature. In addition, the combination of NF and SCF significantly reduced granule size, water solubility, viscosity, and rheological properties with increasing fertilization levels. This study can guide the cultivation of buckwheat with the desired physicochemical properties and provide information for buckwheat-based products in the food industry.

Genome-wide association analysis and transgenic characterization for amylose content regulating gene in tuber of Dioscorea zingiberensis.

BACKGROUND: Amylose, a prebiotic found in yams is known to be beneficial for the gut microflora and is particularly advantageous for diabetic patients' diet. However, the genetic machinery underlying amylose production remains elusive. A comprehensive characterization of the genetic basis of amylose content in yam tubers is a prerequisite for accelerating the genetic engineering of yams with respect to amylose content variation. RESULTS: To uncover the genetic variants underlying variation in amylose content, we evaluated amylose content in freshly harvested tubers from 150 accessions of Dioscorea zingibensis. With 30,000 high-quality single nucleotide polymorphisms (SNP), we performed a genome-wide association analysis (GWAS). The population structure analysis classified the D. zingiberensis accessions into three groups. A total of 115 significant loci were detected on four chromosomes. Of these, 112 significant SNPs (log10(p) = 5, q-value < 0.004) were clustered in a narrow window on the chromosome 6 (chr6). The peak SNP at the position 75,609,202 on chr6 could explain 63.15% of amylose variation in the population and fell into the first exon of the ADP-glucose pyrophosphorylase (AGPase) small subunit gene, causing a non-synonymous modification of the resulting protein sequence. Allele segregation analysis showed that accessions with the rare G allele had a higher amylose content than those harboring the common A allele. However, AGPase, a key enzyme precursor of amylose biosynthesis, was not expressed differentially between accessions with A and G alleles. Overexpression of the two variants of AGPase in Arabidopsis thaliana resulted in a significantly higher amylose content in lines transformed with the AGPase-G allele. CONCLUSIONS: Overall, this study showed that a major genetic variant in AGPase probably enhances the enzyme activity leading to high amylose content in D. zingiberensis tuber. The results provide valuable insights for the development of amylose-enriched genotypes.

Multi-scale structural insights on starch digestibility of instant rice.

Multi-scale structures were investigated to understand starch digestibility of instant rice. A wide range of maximum starch digested ratio, up to about 20%, was observed among instant rice prepared from different rice varieties. Instant rice with a smooth and densely packed cross-section showed slower starch digestibility than those with a porous and loosely packed structure. All samples displayed B + V type crystallinity, with V-type crystallinity negatively correlating with maximum starch digested percentage. After digestion, starch chain-length distributions were significantly altered: rapidly digested starch comprised long amylose and short amylopectin chains, while slowly digested starch comprised chains with a peak degree of polymerization (DP) around 130. These results indicate that instant rice with a compact microstructure, high V-type crystallinity, and DP 130 fractions during digestion can reduce starch digestibility. This study provides insights for food industry to develop instant rice products with slow starch digestibility, potentially improving human health.

The Waxy Gene Has Pleiotropic Effects on Hot Water-Soluble and -Insoluble Amylose Contents in Rice (Oryza sativa) Grains.

Rice (Oryza sativa) is a cereal crop with a starchy endosperm. Starch is composed of amylose and amylopectin. Amylose content (AC) is the principal determinant of rice quality, but varieties with similar ACs can still vary substantially in their quality. In this study, we analyzed the total AC (TAC) and its constituent fractions, the hot water-soluble amylose content (SAC) and hot water-insoluble amylose content (IAC), in two sets of related chromosome segment substitution lines of rice with a common genetic background grown in two years. We searched for quantitative trait loci (QTLs) associated with SAC, IAC, and TAC and identified one common QTL (qSAC-6, qIAC-6, and qTAC-6) on chromosome 6. Map-based cloning revealed that the gene underlying the trait associated with this common QTL is Waxy (Wx). An analysis of the colors of soluble and insoluble starch-iodine complexes and their λmax values (wavelengths at the positions of their peak absorbance values) as well as gel permeation chromatography revealed that Wx is responsible for the biosynthesis of amylose, comprising a large proportion of the soluble fractions of the SAC. Wx is also involved in the biosynthesis of long chains of amylopectin, comprising the hot water-insoluble fractions of the IAC. These findings highlight the pleiotropic effects of Wx on the SAC and IAC. This pleiotropy indicates that these traits have a positive genetic correlation. Therefore, further studies of rice quality should use rice varieties with the same Wx genotype to eliminate the pleiotropic effects of this gene, allowing the independent relationship between the SAC or IAC and rice quality to be elucidated through a multiple correlation analysis. These findings are applicable to other valuable cereal crops as well.

ABA-mediated regulation of rice grain quality and seed dormancy via the NF-YB1-SLRL2-bHLH144 Module.

Abscisic acid (ABA) plays a crucial role in promoting plant stress resistance and seed dormancy. However, how ABA regulates rice quality remains unclear. This study identifies a key transcription factor SLR1-like2 (SLRL2), which mediates the ABA-regulated amylose content (AC) of rice. Mechanistically, SLRL2 interacts with NF-YB1 to co-regulate Wx, a determinant of AC and rice quality. In contrast to SLR1, SLRL2 is ABA inducible but insensitive to GA. In addition, SLRL2 exhibits DNA-binding activity and directly regulates the expression of Wx, bHLH144 and MFT2. SLRL2 competes with NF-YC12 for interaction with NF-YB1. NF-YB1 also directly represses SLRL2 transcription. Genetic validation supports that SLRL2 functions downstream of NF-YB1 and bHLH144 in regulating rice AC. Thus, an NF-YB1-SLRL2-bHLH144 regulatory module is successfully revealed. Furthermore, SLRL2 regulates rice dormancy by modulating the expression of MFT2. In conclusion, this study revealed an ABA-responsive regulatory cascade that functions in both rice quality and seed dormancy.