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Wheat is a staple food for more than 35% of the world's population, with wheat flour used to make hundreds of baked goods. Superior end-use quality is a major breeding target; however, improving it is especially time-consuming and expensive. Furthermore, genes encoding seed-storage proteins (SSPs) form multi-gene families and are repetitive, with gaps commonplace in several genome assemblies. To overcome these barriers and efficiently identify superior wheat SSP alleles, we developed "PanSK" (Pan-SSP k-mer) for genotype-to-phenotype prediction based on an SSP-based pangenome resource. PanSK uses 29-mer sequences that represent each SSP gene at the pangenomic level to reveal untapped diversity across landraces and modern cultivars. Genome-wide association studies with k-mers identified 23 SSP genes associated with end-use quality that represent novel targets for improvement. We evaluated the effect of rye secalin genes on end-use quality and found that removal of ω-secalins from 1BL/1RS wheat translocation lines is associated with enhanced end-use quality. Finally, using machine-learning-based prediction inspired by PanSK, we predicted the quality phenotypes with high accuracy from genotypes alone. This study provides an effective approach for genome design based on SSP genes, enabling the breeding of wheat varieties with superior processing capabilities and improved end-use quality., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

Key Message: This study precisely mapped and validated a quantitative trait locus (QTL) located on chromosome 4B for flag leaf angle in wheat. Flag leaf angle (FLANG) is closely related to crop architecture and yield. We previously identified the quantitative trait locus (QTL) QFLANG-4B for FLANG on chromosome 4B, located within a 14-cM interval flanked by the markers Xbarc20 and Xzyh357, using a mapping population of recombinant inbred lines (RILs) derived from a cross between Nongda3331 (ND3331) and Zang1817. In this study, we fine-mapped QFLANG-4B and validated its associated genetic effect. We developed a BC 3 F 3 population using ND3331 as the recurrent parent through marker-assisted selection, as well as near-isogenic lines (NILs) by selfing BC 3 F 3 plants carrying different heterozygous segments for the QFLANG-4B region. We obtained eight recombinant types for QFLANG-4B, narrowing its location down to a 5.3-Mb region. This region contained 76 predicted genes, 7 of which we considered to be likely candidate genes for QFLANG-4B. Marker and phenotypic analyses of individual plants from the secondary mapping populations and their progeny revealed that the FLANG of the ND3331 allele is significantly higher than that of the Zang1817 allele in multiple environments. These results not only provide a basis for the map-based cloning of QFLANG-4B, but also indicate that QFLANG-4B has great potential for marker-assisted selection in wheat breeding programs designed to improve plant architecture and yield., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Key Message: A point mutation of RPM1 triggers persistent immune response that induces leaf premature senescence in wheat, providing novel information of immune responses and leaf senescence. Leaf premature senescence in wheat (Triticum aestivum L.) is one of the most common factors affecting the plant's development and yield. In this study, we identified a novel wheat mutant, yellow leaf and premature senescence (ylp), which exhibits yellow leaves and premature senescence at the heading and flowering stages. Consistent with the yellow leaves phenotype, ylp had damaged and collapsed chloroplasts. Map-based cloning revealed that the phenotype of ylp was caused by a point mutation from Arg to His at amino acid 790 in a plasma membrane-localized protein resistance to Pseudomonas syringae pv. maculicola 1 (RPM1). The point mutation triggered excessive immune responses and the upregulation of senescence- and autophagy-associated genes. This work provided the information for understanding the molecular regulatory mechanism of leaf senescence, and the results would be important to analyze which mutations of RPM1 could enable plants to obtain immune activation without negative effects on plant growth., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Gluten is composed of glutenins and gliadins and determines the viscoelastic properties of dough and end-use quality in wheat (Triticum aestivum L.). Gliadins are important for wheat end-use traits, but the contribution of individual gliadin genes is unclear, since gliadins are encoded by a complex, multigenic family, including many pseudogenes. We used CRISPR/Cas9-mediated gene editing and map-based cloning to investigate the contribution of the γ-gliadin genes annotated in the wheat cultivar 'Fielder', showing that Gli-γ1-1D and Gli-γ2-1B account for most of the γ-gliadin accumulation. The impaired activity of only two γ-gliadin genes in knockout mutants improved end-use quality and reduced gluten epitopes associated with celiac disease (CD). Furthermore, we identified an elite haplotype of Gli-γ1-1D linked to higher end-use quality in a wheat germplasm collection and developed a molecular marker for this allele for marker-assisted selection. Our findings provide information and tools for biotechnology-based and classical breeding programs aimed at improving wheat end-use quality., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Key Message: We identified ten QTLs controlling SDS-SV trait in a RIL population derived from ND3331 and Zang1817. Pinb-D1p is an elite allele from Tibetan semi‑wild wheat for good end-use quality. Gluten strength is an important factor for wheat processing and end-product quality and is commonly characterized using the sodium dodecyl sulfate-sedimentation volume (SDS-SV) test. The objective of this study was to identify quantitative trait loci (QTLs) associated with wheat SDS-SV traits using a recombinant inbred line (RIL) population derived from common wheat line NongDa3331 (ND3331) and Tibetan semi-wild wheat accession Zang1817. We detected 10 QTLs controlling SDS-SV on chromosomes 1A, 1B, 3A, 4A, 4B, 5A, 5D, 6B and 7A, with individual QTLs explaining 2.02% to 15.53% of the phenotypic variation. They included four major QTLs, Qsdss-1A, Qsdss-1B.1, Qsdss-1B.2, and Qsdss-5D, whose effects on SDS-SV were due to the Glu-A1 locus encoding the high-molecular-weight glutenin subunit 1Ax1, the 1B/1R translocation, 1Bx7 + 1By8 at the Glu-B1 locus, and the hardness-controlling loci Pina-D1 and Pinb-D1, respectively. We developed KASP markers for the Glu-A1, Glu-B1, and Pinb-D1 loci. Importantly, we showed for the first time that the hardness allele Pinb-D1p positively affects SDS-SV, making it a good candidate for wheat quality improvement. These results broaden our understanding of the genetic characterization of SDS-SV, and the QTLs identified are potential target regions for fine-mapping and marker-assisted selection in wheat breeding programs., (© 2022. The Author(s).)

Along with increasing demands for high yield, elite processing quality and improved nutrient value in wheat, concerns have emerged around the effects of gluten in wheat-based foods on human health. However, knowledge of the mechanisms regulating gluten accumulation remains largely unexplored. Here we report the identification and characterization of a wheat low gluten protein 1 (lgp1) mutant that shows extremely low levels of gliadins and glutenins. The lgp1 mutation in a single γ-gliadin gene causes defective signal peptide cleavage, resulting in the accumulation of an excessive amount of unprocessed γ-gliadin and a reduced level of gluten, which alters the endoplasmic reticulum (ER) structure, forms the autophagosome-like structures, leads to the delivery of seed storage proteins to the extracellular space and causes a reduction in starch biosynthesis. Physiologically, these effects trigger ER stress and cell death. This study unravels a unique mechanism that unprocessed γ-gliadin reduces gluten accumulation associated with ER stress and elevated cell death in wheat. Moreover, the reduced gluten level in the lgp1 mutant makes it a good candidate for specific diets for patients with diabetes or kidney diease., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Main Conclusion: The function of SQUAMOSA PROMOTER-BINDING PROTEIN-BOX gene TaSPL14 in wheat is similar to that of OsSPL14 in rice in regulating plant height, panicle length, spikelet number, and thousand-grain weight of wheat, but differs during tiller development. TaSPL14 may regulate spike development via ethylene-response gene EIN3-LIKE 1 (TaEIL1), ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR 2.11 (TaRAP2.11), and ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR 1 (TaERF1), but not DENSE AND ERECT PANICLE 1 (TaDEP1) in wheat. The SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE gene OsSPL14 from rice is considered to be a major determinant of ideal plant architecture consisting of few unproductive tillers, more grains per spike, and high resistance of stems to lodging. However, the function of its orthologous gene, TaSPL14, in wheat is unknown. Here, we reported the functional similarities and differences between TaSPL14 and OsSPL14. Similar to OsSPL14 knock-outs in rice, wheat TaSPL14 knock-out plants exhibited decreased plant height, panicle length, spikelet number, and thousand-grain weight. In contrast to OsSPL14, however, TaSPL14 did not affect tiller number. Transcriptome analysis revealed that the expression of genes related to ethylene response was significantly decreased in young spikes of TaSPL14 knock-out lines as compared with wild type. TaSPL14 directly binds to the promoters of the ethylene-response genes TaEIL1, TaRAP2.11, and TaERF1, and promotes their expression, suggesting that TaSPL14 might regulate wheat spike development via the ethylene-response pathway. The elucidation of TaSPL14 will contribute to understanding of the molecular mechanisms that underlie wheat plant architecture.

Protein ubiquitination is a major post-translational modification important for diverse biological processes. In wheat (Triticum aestivum) and Arabidopsis thaliana, STRESS-ASSOCIATED PROTEIN 5 (SAP5) is involved in drought tolerance, acting as an E3 ubiquitin ligase to target DRIP and MBP-1 for degradation. To identify further target proteins of SAP5, we implemented two independent approaches in this work. We used ubiquitylome capture with a di-Gly-Lys antibody-based peptide enrichment and affinity purification with a polyubiquitin antibody coupled with mass spectrometry to elucidate the SAP5-dependent ubiquitylation of its target proteins in response to osmotic stress. Wild type or TaSAP5-overexpressing Arabidopsis line, which was more tolerant to osmotic stress according to our previous study, were used here. We identified HSP90C (chloroplast heat shock protein 90) as a substrate of TaSAP5. Further biochemical experiments indicated that TaSAP5 interacts with HSP90C and mediates its degradation by the 26S proteasome. Our work also demonstrates that ubiquitylome profiling is an effective approach to search for substrates of the TaSAP5 E3 ubiquitin ligase when heterologously expressed in Arabidopsis., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

In grass crops, leaf angle is determined by development of the lamina joint, the tissue connecting the leaf blade and sheath, and is closely related to crop architecture and yield. In this study, we identified a mutant generated by fast neutron radiation that exhibited an erect leaf phenotype caused by defects in lamina joint development. Map-based cloning revealed that the gene TaSPL8 , encoding a SQUAMOSA PROMOTER BINDING-LIKE (SPL) protein, is deleted in this mutant. TaSPL8 knock-out mutants exhibit erect leaves due to loss of the lamina joint, compact architecture, and increased spike number especially in high planting density, suggesting similarity with its LIGULESS1 homologs in maize ( Zea mays ) and rice ( Oryza sativa ). Hence, LG1 could be a robust target for plant architecture improvement in grass species. Common wheat ( Triticum aestivum , 2 n = 6× = 42; BBAADD) is an allohexaploid containing A, B, and D subgenomes and the homeologous gene of TaSPL8 from the D subgenome contributes to the length of the lamina joint to a greater extent than that from the A and B subgenomes. Comparison of the transcriptome between the Taspl8 mutant and the wild type revealed that TaSPL8 is involved in the activation of genes related to auxin and brassinosteroid pathways and cell elongation. TaSPL8 binds to the promoters of the AUXIN RESPONSE FACTOR gene and of the brassinosteroid biogenesis gene CYP90D2 and activates their expression. These results indicate that TaSPL8 might regulate lamina joint development through auxin signaling and the brassinosteroid biosynthesis pathway., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Key Message: QTL controlling flag leaf length, flag leaf width, flag leaf area and flag leaf angle were mapped in wheat. This study aimed to advance our understanding of the genetic mechanisms underlying morphological traits of the flag leaves of wheat (Triticum aestivum L.). A recombinant inbred line (RIL) population derived from ND3331 and the Tibetan semi-wild wheat Zang1817 was used to identify quantitative trait loci (QTLs) controlling flag leaf length (FLL), flag leaf width (FLW), flag leaf area (FLA), and flag leaf angle (FLANG). Using an available simple sequence repeat genetic linkage map, 23 putative QTLs for FLL, FLW, FLA, and FLANG were detected on chromosomes 1B, 2B, 3A, 3D, 4B, 5A, 6B, 7B, and 7D. Individual QTL explained 4.3-68.52% of the phenotypic variance in different environments. Four QTLs for FLL, two for FLW, four for FLA, and five for FLANG were detected in at least two environments. Positive alleles of 17 QTLs for flag leaf-related traits originated from ND3331 and 6 originated from Zang1817. QTLs with pleiotropic effects or multiple linked QTL were also identified on chromosomes 1B, 4B, and 5A; these are potential target regions for fine-mapping and marker-assisted selection in wheat breeding programs.

Seed germination is important for grain yield and quality and rapid, near-simultaneous germination helps in cultivation; however, cultivars that germinate too readily can undergo preharvest sprouting (PHS), which causes substantial losses in areas that tend to get rain around harvest time. Moreover, our knowledge of mechanisms regulating seed germination in wheat ( Triticum aestivum ) remains limited. In this study, we analyzed function of a wheat-specific microRNA 9678 (miR9678), which is specifically expressed in the scutellum of developing and germinating seeds. Overexpression of miR9678 delayed germination and improved resistance to PHS in wheat through reducing bioactive gibberellin (GA) levels; miR9678 silencing enhanced germination rates. We provide evidence that miR9678 targets a long noncoding RNA ( WSGAR ) and triggers the generation of phased small interfering RNAs that play a role in the delay of seed germination. Finally, we found that abscisic acid (ABA) signaling proteins bind the promoter of miR9678 precursor and activate its expression, indicating that miR9678 affects germination by modulating the GA/ABA signaling., (© 2018 American Society of Plant Biologists. All rights reserved.)

Heat stress poses a serious threat to global crop production. In efforts that aim to mitigate the adverse effects of heat stress on crops, a variety of genetic tools are being used to develop plants with improved thermotolerance. The characterization of important regulators of heat stress tolerance provides essential information for this aim. In this study, we examine the wheat (Triticum aestivum) NAC transcription factor gene TaNAC2L. High temperature induced TaNAC2L expression in wheat and overexpression of TaNAC2L in Arabidopsis thaliana enhanced acquired heat tolerance without causing obvious alterations in phenotype compared with wild type under normal conditions. TaNAC2L overexpression also activated the expression of heat-related genes in the transgenic Arabidopsis plants, suggesting that TaNAC2L may improve heat tolerance by regulating the expression of stress-responsive genes. Notably, TaNAC2L is also regulated at the post-translational level and might be degraded via a proteasome-mediated pathway. Thus, this wheat transcription factor may have potential uses in enhancing thermotolerance in crops.

Background: MicroRNAs (miRNAs) are small, non-coding RNAs playing essential roles in plant growth, development, and stress responses. Sequencing of small RNAs is a starting point for understanding their number, diversity, expression and possible roles in plants., Results: In this study, we conducted a genome-wide survey of wheat miRNAs from 11 tissues, characterizing a total of 323 novel miRNAs belonging to 276 families in wheat. A miRNA conservation analysis identified 191 wheat-specific miRNAs, 2 monocot-specific miRNAs, and 30 wheat-specific variants from 9 highly conserved miRNA families. To understand possible roles of wheat miRNAs, we determined 524 potential targets for 124 miRNA families through degradome sequencing, and cleavage of a subset of them was validated via 5' RACE. Based on the genome-wide identification and characterization of miRNAs and their associated target genes, we further identified 64 miRNAs preferentially expressing in developing or germinating grains, which could play important roles in grain development., Conclusion: We discovered 323 wheat novel miRNAs and 524 target genes for 124 miRNA families in a genome-wide level, and our data will serve as a foundation for future research into the functional roles of miRNAs in wheat.

Small RNAs related to RNA interference are key molecules in many developmental processes, in which they can both regulate developmental gene expression and maintain the integrity of the genome and epigenome. In plants, short interfering RNAs (siRNAs) of 24 nt in length are an abundant type of small RNA associated with transposable elements (TEs), other repetitive sequences, and viral defense. One means by which TE-associated siRNAs affect genome integrity is by altering chromatin structure through a process called RNA-directed DNA methylation (RdDM). In this paper, we describe a comparative survey of siRNAs from wheat seedling leaves, seedling roots, young spikelets, and grains at 8 and 15 days after pollination (DAP). We find that the general patterns of siRNA distributions are similar across different TEs and within TEs of the same family regardless of tissue, but the relative abundance of 24-nt siRNAs is highest in developing grains. We also find that TEs that are transcriptionally active in endosperm are associated with the highest siRNA abundance not only in grains, but also in other tissues as well. These results suggest that RdDM is an important feature of developing wheat grain and are consistent with the hypothesis that TE expression in endosperm results in increased TE siRNAs, and that RdDM is a conserved feature of plant seed development., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

Differential gene expression between hybrids and their parents is considered to be associated with heterosis. However, the physiological functions and possible contribution to heterosis of these differentially expressed genes are unknown. We have isolated one hybrid upregulated gene encoding putative wheat ADP-ribosylation factor, designated TaARF. In this study, real-time quantitative reverse transcription-polymerase chain reaction analysis indicated that the TaARF transcript was preferentially expressed in root, node and crown, and the accumulation of TaARF mRNA in hybrid was more than 1.5-fold higher than that in two parents. In order to understand possible roles of the putative wheat ARF gene, TaARF was overexpressed in Arabidopsis, and the transgenic plants were characterized. We show that ectopic overexpression of TaARF in Arabidopsis leads to increased leaf area, increased growth rate and earlier transition to flowering, suggesting that TaARF plays significant roles in growth and development. This study provides evidence demonstrating that TaARF plays important roles in growth and development and we speculate that the upregulated expression of this gene might contribute to the heterosis observed in wheat root and leaf growth.

Background: MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition. So far, identification of miRNAs has been limited to a few model plant species, such as Arabidopsis, rice and Populus, whose genomes have been sequenced. Wheat is one of the most important cereal crops worldwide. To date, only a few conserved miRNAs have been predicted in wheat and the computational identification of wheat miRNAs requires the genome sequence, which is unknown., Results: To identify novel as well as conserved miRNAs in wheat (Triticum aestivum L.), we constructed a small RNA library. High throughput sequencing of the library and subsequent analysis revealed the identification of 58 miRNAs, comprising 43 miRNA families. Of these, 35 miRNAs belong to 20 conserved miRNA families. The remaining 23 miRNAs are novel and form 23 miRNA families in wheat; more importantly, 4 of these new miRNAs (miR506, miR510, miR514 and miR516) appear to be monocot-specific. Northern blot analysis indicated that some of the new miRNAs are preferentially expressed in certain tissues. Based on sequence homology, we predicted 46 potential targets. Thus, we have identified a large number of monocot-specific and wheat-specific miRNAs. These results indicate that both conserved and wheat-specific miRNAs play important roles in wheat growth and development, stress responses and other physiological processes., Conclusion: This study led to the discovery of 58 wheat miRNAs comprising 43 miRNA families; 20 of these families are conserved and 23 are novel in wheat. It provides a first large scale cloning and characterization of wheat miRNAs and their predicted targets.