Regulation of Amylose Content by Single Mutations at an Active Site in the Wx-B1 Gene in a Tetraploid Wheat Mutant.

The granule-bound starch synthase I (GBSSI) encoded by the waxy gene is responsible for amylose synthesis in the endosperm of wheat grains. In the present study, a novel Wx-B1 null mutant line, M3-415, was identified from an ethyl methanesulfonate-mutagenized population of Chinese tetraploid wheat landrace Jianyangailanmai (LM47). The gene sequence indicated that the mutated Wx-B1 encoded a complete protein; this protein was incompatible with the protein profile obtained using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed the lack of Wx-B1 protein in the mutant line. The prediction of the protein structure showed an amino acid substitution (G470D) at the edge of the ADPG binding pocket, which might affect the binding of Wx-B1 to starch granules. Site-directed mutagenesis was further performed to artificially change the amino acid at the sequence position 469 from alanine (A) to threonine (T) (A469T) downstream of the mutated site in M3-415. Our results indicated that a single amino acid mutation in Wx-B1 reduces its activity by impairing its starch-binding capacity. The present study is the first to report the novel mechanism underlying Wx-1 deletion in wheat; moreover, it provided new insights into the inactivation of the waxy gene and revealed that fine regulation of wheat amylose content is possible by modifying the GBSSI activity.

Analysis of starch structure and functional properties of tetraploid wheat (Triticum turgidum L.) with differing waxy protein composition.

BACKGROUND: An increased demand for food has mirrored the increasing global population. Obesity and diabetes are two disorders induced by poor eating choices. Consequently, there is an urgent need to develop modified foods that can ameliorate such illnesses. The objective of this study was to explore the effect of Waxy genes on the structural and functional properties of starch, with the aim of improving food quality. Wild-type tetraploid wheat was compared with three mutants with different Waxy gene combinations. RESULTS: The proportion of B-type granules was higher in the mutants than in the wild-type (Wx-AB), and there were significant changes in the starch granule size, number, and phenotype in the Wx free mutant (Wx-ab). The lowest branch chain length was observed in Wx-ab, whereas Wx-AB had the highest branch chain length of DP ≥ 37. Wx-ab had the highest degree of crystallinity. The crystallinity trend followed the order Wx-ab>Wx-Ab>Wx-aB>Wx-AB. The amount of slowly digestible starch (SDS) was higher in native, gelatinized, and retrograded starch in the mutant. The amount of retrograded starch was closer to gelatinized starch than to native starch. CONCLUSION: Waxy proteins make a substantial contribution to starch structure. A lack of waxy proteins reduced the unit chains markedly compared with the control. Waxy proteins significantly affected the smaller and longer chains of starch. The lines with differing waxy composition had different effects on food digestion. The Wx-AB in native starch and Wx-Ab in gelatinized starch can control obesity and diabetes by slow-digesting carbohydrates and high resistance to digestion. © 2022 Society of Chemical Industry.

A single base change at exon of Wx-A1 caused gene inactivation and starch properties modified in a wheat EMS mutant line.

BACKGROUND: Wheat is an essential source of starch. The GBSS or waxy genes are responsible for synthesizing amylose in cereals. The present study identified a novel Wx-A1 null mutant line from an ethyl methanesulfonate (EMS)-mutagenized population of common wheat cv. SM126 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose gel analyses. RESULTS: The alignment of the Wx-A1 gene sequences from the mutant and parental SM126 lines showed only one single nucleotide polymorphism causing the appearance of a premature stop codon and Wx-A1 inactivation. The lack of Wx-A1 protein resulted in decreased amylose, total starch and resistant starch. The starch morphology assessment revealed that starch from mutant seeds was more wrinkled, increasing its susceptibility to digestion. Regarding the starch thermodynamic properties, the gelatinization temperature was remarkably reduced in the mutant compared to parental line SM126. The digestibility of native, gelatinized, and retrograded starches was analyzed for mutant M4-627 and the parental SM126 line. In the M4-627 line, rapidly digestible starch contents were increased, whereas resistant starch was decreased in the three types of starch. CONCLUSION: Waxy protein is essential for starch synthesis. The thermodynamic characteristics were decreased in the Wx-A1 mutant line. The digestibility properties of starch were also affected. Therefore, the partial waxy mutant M3-627 might play a significant role in food improvement. Furthermore, it might also be used to produce high-quality noodles. © 2021 Society of Chemical Industry.

The production of wheat - Aegilops sharonensis 1Ssh chromosome substitution lines harboring alien novel high-molecular-weight glutenin subunits.

In our previous work, a novel high-molecular-weight glutenin subunit (HMW-GS) with an extremely large molecular weight from Aegilops sharonensis was identified that may contribute to excellent wheat (Triticum aestivum) processing quality and increased dough strength, and we further generated HMW-GS homozygous lines by crossing. In this study, we crossed the HMW-GS homozygous line 66-17-52 with 'Chinese Spring' Ph1 mutant CS ph1b to induce chromosome recombination between wheat and Ae. sharonensis. SDS-PAGE was used to identify 19 derived F2 lines with the HMW-GSs of Ae sharonensis. The results of non-denaturing fluorescence in situ hybridization (ND-FISH) indicated that lines 6-1 and 6-7 possessed a substitution of both 5D chromosomes by a pair of 1Ssh chromosomes. Further verification by newly developed 1Ssh-specific chromosome markers showed that these two lines amplified the expected fragment. Thus, it was concluded that lines 6-1 and 6-7 are 1Ssh(5D) chromosome substitution lines. The 1Ssh(5D) chromosome substitution lines, possessing alien subunits with satisfactory quality-associated structural features of large repetitive domains and increased number of subunits, may have great potential in strengthening the viscosity and elasticity of dough made from wheat flour. Therefore, these substitution lines can be used for wheat quality improvement and further production of 1Ssh translocation lines.

HvGBSSI mutation at the splicing receptor site affected RNA splicing and decreased amylose content in barley.

Granule-bound starch synthase I (HvGBSSI) is encoded by the barley waxy (Wx-1) gene and is the sole enzyme in the synthesis of amylose. Here, a Wx-1 mutant was identified from an ethyl methane sulfonate (EMS)-mutagenized barley population. There were two single-base mutations G1086A and A2424G in Wx-1 in the mutant (M2-1105). The G1086A mutation is located at the 3' splicing receptor (AG) site of the fourth intron, resulting in an abnormal RNA splicing. The A2424G mutation was a synonymous mutation in the ninth intron. The pre-mRNA of Wx-1 was incorrectly spliced and transcribed into two abnormal transcripts. The type I transcript had a 6 bp deletion in the 5' of fifth exon, leading to a translated HvGBSSI protein lacking two amino acids with a decreased starch-binding capacity. In the type II transcript, the fourth intron was incorrectly cleaved and retained, resulting in the premature termination of the barley Wx-1 gene. The mutations in the Wx-1 decreased the enzymatic activity of the HvGBSSI enzyme and resulted in a decreased level in amylose content. This work sheds light on a new Wx-1 gene inaction mechanism.

Re-examination of the APETALA2/Ethylene-Responsive Factor Gene Family in Barley (Hordeum vulgare L.) Indicates a Role in the Regulation of Starch Synthesis.

The APETALA2/Ethylene-Responsive factor (AP2/ERF) gene family is a large plant-specific transcription factor family, which plays important roles in regulating plant growth and development. A role in starch synthesis is among the multiple functions of this family of transcription factors. Barley (Hordeum vulgare L.) is one of the most important cereals for starch production. However, there are limited data on the contribution of AP2 transcription factors in barley. In this study, we used the recently published barley genome database (Morex) to identify 185 genes of the HvAP2/ERF family. Compared with previous work, we identified 64 new genes in the HvAP2/ERF gene family and corrected some previously misannotated and duplicated genes. After phylogenetic analysis, HvAP2/ERF genes were classified into four subfamilies and 18 subgroups. Expression profiling showed different patterns of spatial and temporal expression for HvAP2/ERF genes. Most of the 12 HvAP2/ERF genes analyzed using quantitative reverse transcription-polymerase chain reaction had similar expression patterns when compared with those of starch synthase genes in barley, except for HvAP2-18 and HvERF-73. HvAP2-18 is homologous to OsRSR1, which negatively regulates the synthesis of rice starch. Luciferase reporter gene, and yeast one-hybrid assays showed that HvAP2-18 bound the promoter of AGP-S and SBE1 in vitro. Thus, HvAP2-18 might be an interesting candidate gene to further explore the mechanisms involved in the regulation of starch synthesis in barley.

Optimal Nitrogen Fertilization Management of Seed-sowing Rapeseed in Yangtze River Basin of China.

BACKGROUND AND OBJECTIVE: For cultivation and high yield of oilseed rape (Brassica napus L.) in China, traditional seedling transplanting is replaced by seed-sowing but, better nitrogen management is crucial and not established yet. This study aimed to adapt N management to the seed-sowing method for the winter oilseed rape and to minimize the N fertilizer-derived pollution potential in the upper reaches of Yangtze River Basin. MATERIALS AND METHODS: Three field experiments were conducted to check effect of different doses of N fertilizers, split doses of N and different types of N fertilizers for seed-sowing winter oilseed rape with high plant density in upper reaches of Yangtze River Basin in Sichuan province of China. RESULTS: In first experiment, among four doses (0, 90, 180 and 270 kg N ha-1) on average 3.54 t ha-1 was in 180 kg N ha-1 and 3.61 t ha-1 in 270 kg N ha-1 while cultivars dy6 and cn3 produced 3.23 and 3.29 t ha-1 which is significantly higher than zs11. There was no significant difference in N-use efficiency among three cultivars tested and second experiment showed no significant difference in seed yield with split N application. The third experiment compared the effects of different fertilizer types (urea, coated urea, 1:1 mixture of urea and coated urea and compound nitrogen fertilizer) on seed yield and get no significant difference in seed yield. CONCLUSION: This experiment proved that seed sowing method with higher nitrogen had high yield in the upper reaches of Yangtze River Basin in China, but higher N application may cause environment pollution. So, seed sowing method with nitrogen 180 kg N ha-1 was proved to be more effective.

The inter- and intra-rater reliability of the Maestro and Barroco metatarsal length measurement techniques.

BACKGROUND: The relationship between metatarsal length and various forefoot pathologies is a topic of contention in Orthopaedics. The results of such investigations have been shown to depend on the method of metatarsal length measurement used. The aim of this study was to assess the inter- and intra-rater reliability of the Maestro and Barroco metatarsal length measurement techniques. METHODS: A retrospective and quantitative study was performed on 15 randomly selected radiographs to determine the reliability of the two measurement techniques across all five metatarsals (M1 to M5). This was done at one week apart for three weeks by three raters. The intraclass correlation coefficient (ICC), and the 95% lower confidence limit (95% LCL) were calculated. RESULTS: The Maestro and Barroco techniques produced high to very high ICC vlaues for length measurements across all metatarsals. The 95% lower confidence limit for inter-rater measurements ranged between 0.92-0.98 for Maestro's and 0.86-0.99 for Barroco's technique. For intra-rater measurements the 95% LCL ranged between 0.83-0.99 for Maestro's and 0.75-0.99 for Barroco's technique. CONCLUSIONS: Our study found that both the Maestro and Barroco methods of measurements produced high to very high inter- and intra-rater reliability. Both methods may be suitable for the use of peri-operative planning and clinical research relating metatarsal length and forefoot pathology. Besides having a more simplistic method of application, the novel Barroco technique is comparable to the more established Maestro method in both repeatability and reproducibility.