Discovery of a novel flavonol O-methyltransferase possessing sequential 4'- and 7-O-methyltransferase activity from Camptotheca acuminata Decne.

The biosynthetic route for flavonol in Camptotheca acuminata has been recently elucidated from a chemical point of view. However, the genes involved in flavonol methylation remain unclear. It is a critical step for fully uncovering the flavonol metabolism in this ancient plant. In this study, the multi-omics resource of this plant was utilized to perform flavonol O-methyltransferase-oriented mining and screening. Two genes, CaFOMT1 and CaFOMT2 are identified, and their recombinant CaFOMT proteins are purified to homogeneity. CaFOMT1 exhibits strict substrate and catalytic position specificity for quercetin, and selectively methylates only the 4'-OH group. CaFOMT2 possesses sequential O-methyltransferase activity for the 4'-OH and 7-OH of quercetin. These CaFOMT genes are enriched in the leaf and root tissues. The catalytic dyad and critical substrate-binding sites of the CaFOMTs are determined by molecular docking and further verified through site-mutation experiments. PHE181 and MET185 are designated as the critical sites for flavonol substrate selectivity. Genomic environment analysis indicates that CaFOMTs evolved independently and that their ancestral genes are different from that of the known Ca10OMT. This study provides molecular insights into the substrate-binding pockets of two new CaFOMTs responsible for flavonol metabolism in C. acuminata.

Identification and fine-mapping of QYrAS286-2BL conferring adult-plant resistance to stripe rust in cultivated emmer wheat.

KEY MESSAGE: A novel major adult-plant stripe rust resistance QTL derived from cultivated emmer wheat was mapped to a 123.6-kb region on wheat chromosome 2BL. Stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici (Pst), is one of the most devastating diseases of wheat. Identification of new sources of resistance and their utilization in breeding programs is the effectively control strategy. The objective of this study was to identify and genetically characterize the stripe rust resistance derived from the cultivated emmer accession AS286. A recombinant inbred line population, developed from a cross between the susceptible durum wheat line langdon and AS286, was genotyped using the Wheat55K single nucleotide polymorphism array and evaluated in field conditions with a mixture of the prevalent Chinese Pst races (CYR32, CYR33, CYR34, Zhong4, and HY46) and in growth chamber with race CYR34. Three QTLs conferring resistance were mapped on chromosomes 1BS, 2BL, and 5BL, respectively. The QYrAS286-1BS and QYrAS286-2BL were stable with major effects, explaining 12.91% to 18.82% and 11.31% to 31.43% of phenotypic variation, respectively. QYrAS286-5BL was only detected based on growth chamber seedling data. RILs harboring both QYrAS286-1BS and QYrAS286-2BL showed high levels of stripe rust resistance equal to the parent AS286. The QYrAS286-2BL was only detected at the adult-plant stage, which is different from previously named Yr genes and inherited as a single gene. It was further mapped to a 123.6-kb region using KASP markers derived from SNPs identified by bulked segregant RNA sequencing (BSR-Seq). The identified loci enrich our stripe rust resistance gene pool, and the flanking markers developed here could be useful in marker-assisted selection for incorporating QYrAS286-2BL into wheat cultivars.

Genome-Wide Survey and Functional Verification of the NAC Transcription Factor Family in Wild Emmer Wheat.

The NAC transcription factor (TF) family is one of the largest TF families in plants, which has been widely reported in rice, maize and common wheat. However, the significance of the NAC TF family in wild emmer wheat (Triticum turgidum ssp. dicoccoides) is not yet well understood. In this study, a genome-wide investigation of NAC genes was conducted in the wild emmer genome and 249 NAC family members (TdNACs) were identified. The results showed that all of these genes contained NAM/NAC-conserved domains and most of them were predicted to be located on the nucleus. Phylogenetic analysis showed that these 249 TdNACs can be classified into seven clades, which are likely to be involved in the regulation of grain protein content, starch synthesis and response to biotic and abiotic stresses. Expression pattern analysis revealed that TdNACs were highly expressed in different wheat tissues such as grain, root, leaves and shoots. We found that TdNAC8470 was phylogenetically close to NAC genes that regulate either grain protein or starch accumulation. Overexpression of TdNAC8470 in rice showed increased grain starch concentration but decreased grain Fe, Zn and Mn contents compared with wild-type plants. Protein interaction analysis indicated that TdNAC8470 might interact with granule-bound starch synthase 1 (TdGBSS1) to regulate grain starch accumulation. Our work provides a comprehensive understanding of the NAC TFs family in wild emmer wheat and establishes the way for future functional analysis and genetic improvement of increasing grain starch content in wheat.

Genome-Wide Investigation and Functional Verification of the ZIP Family Transporters in Wild Emmer Wheat.

The zinc/iron-regulated transporter-like protein (ZIP) family has a crucial role in Zn homeostasis of plants. Although the ZIP genes have been systematically studied in many plant species, the significance of this family in wild emmer wheat (Triticum turgidum ssp. dicoccoides) is not yet well understood. In this study, a genome-wide investigation of ZIPs genes based on the wild emmer reference genome was conducted, and 33 TdZIP genes were identified. Protein structure analysis revealed that TdZIP proteins had 1 to 13 transmembrane (TM) domains and most of them were predicted to be located on the plasma membrane. These TdZIPs can be classified into three clades in a phylogenetic tree. They were annotated as being involved in inorganic ion transport and metabolism. Cis-acting analysis showed that several elements were involved in hormone, stresses, grain-filling, and plant development. Expression pattern analysis indicated that TdZIP genes were highly expressed in different tissues. TdZIP genes showed different expression patterns in response to Zn deficiency and that 11 genes were significantly induced in either roots or both roots and shoots of Zn-deficient plants. Yeast complementation analysis showed that TdZIP1A-3, TdZIP6B-1, TdZIP6B-2, TdZIP7A-3, and TdZIP7B-2 have the capacity to transport Zn. Overexpression of TdZIP6B-1 in rice showed increased Zn concentration in roots compared with wild-type plants. The expression levels of TdZIP6B-1 in transgenic rice were upregulated in normal Zn concentration compared to that of no Zn. This work provides a comprehensive understanding of the ZIP gene family in wild emmer wheat and paves the way for future functional analysis and genetic improvement of Zn deficiency tolerance in wheat.

Identification and Mapping of QTL for Stripe Rust Resistance in the Chinese Wheat Cultivar Shumai126.

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a damaging disease of wheat globally, and breeding resistant cultivars is the best control strategy. The Chinese winter wheat cultivar Shumai126 (SM126) exhibited strong resistance to P. striiformis f. sp. tritici in the field for more than 10 years. The objective of this study was to identify and map quantitative trait loci (QTL) for resistance to stripe rust in a population of 154 recombinant inbred lines (RILs) derived from a cross between cultivars Taichang29 (TC29) and SM126. The RILs were tested in six field environments with a mixture of the Chinese prevalent races (CYR32, CYR33, CYR34, Zhong4, and HY46) of P. striiformis f. sp. tritici and in growth chamber with race CYR34 and genotyped using the Wheat55K single nucleotide polymorphism (SNP) array. Six QTL were mapped on chromosomes 1BL, 2AS, 2AL, 6AS, 6BS, and 7BL, respectively. All QTL were contributed by SM126 except QYr.sicau-2AL. The QYr.sicau-1BL and QYr.sicau-2AS had major effects, explaining 27.00 to 39.91% and 11.89 to 17.11% of phenotypic variances, which may correspond to known resistance genes Yr29 and Yr69, respectively. The QYr.sicau-2AL, QYr.sicau-6AS, and QYr.sicau-6BS with minor effects are likely novel. QYr.sicau-7BL was only detected based on growth chamber seedling data. Additive effects were detected for the combination of QYr.sicau-1BL, QYr.sicau-2AS, and QYr.sicau-2AL. SNP markers linked to QYr.sicau-1BL (AX-111056129 and AX-108839316) and QYr.sicau-2AS (AX-111557864 and AX-110433540) were converted to breeder-friendly Kompetitive allele-specific PCR (KASP) markers that would facilitate the deployment of stripe rust resistance genes in wheat breeding.

Comparison of the Agronomic, Cytological, Grain Protein Characteristics, as Well as Transcriptomic Profile of Two Wheat Lines Derived From Wild Emmer.

Two advanced wheat lines BAd7-209 and BAd23-1 without the functional gene GPC-B1 were obtained from a cross between common wheat cultivar Chuannong 16 (CN16) and wild emmer wheat accession D97 (D97). BAd7-209 showed superior quality parameters than those of BAd23-1 and CN16. We found that the components of glutenins and gliadins in BAd7-209 and BAd23-1 were similar, whereas BAd7-209 had higher amount of glutenins and gliadins than those of BAd23-1. RNA sequencing analysis on developing grains of BAd7-209 and BAd23-1 as well as their parents revealed 382 differentially expressed genes (DEGs) between the high-grain protein content (GPC) (D97 + BAd7-209) and the low-GPC (CN16 + BAd23-1) groups. DEGs were mainly associated with transcriptional regulation of the storage protein genes, protein processing in endoplasmic reticulum, and protein export pathways. The upregulated gluten genes and transcription factors (e.g., NAC, MYB, and bZIP) may contribute to the high GPC in BAd7-209. Our results provide insights into the potential regulation pathways underlying wheat grain protein accumulation and contribute to make use of wild emmer for wheat quality improvement.

Transcriptome analysis provides insights into the mechanisms underlying wheat cultivar Shumai126 responding to stripe rust.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a destructive fungal disease of wheat globally. Breeding resistance cultivars is one of the most cost-effective methods to control Pst. Shumai126 (SM126), a high-yielding commercial wheat cultivar, showed strong stripe rust resistance for more than ten years. However, the molecular mechanisms and the responsive genes underlying the SM126 resistance to Pst have not been explored yet. In the present study, RNA-seq was used to analyze changes in the transcriptome at different time points of Pst infection in seedling leaves of SM126. In total, 520, 148 and 1439 differentially expressed genes (DEGs) were found to be up- or down-regulated after Pst infection at 1, 3, and 7 days post inoculation, respectively. The majority of DEGs exhibited transient expression patterns during Pst infection at different time points. GO and KEGG enrichment analysis revealed that many biological processes, such as photosynthesis, flavonoid biosynthesis, oxidative phosphorylation, MAPK signaling pathway, and phenylalanine metabolism are involved in SM126 response to Pst. Expression of genes involved in the plant-pathogen interaction pathway was detected and some key genes showed differential expression. DEGs encoded R proteins and transcription factors were also identified. Our study suggests the gene resources in SM126 related to stripe rust response could be valuable for understanding the mechanisms involved in stripe rust resistance and improvement of wheat resistance to Pst.

Comparative analysis of developing grain transcriptome reveals candidate genes and pathways improving GPC in wheat lines derived from wild emmer.

The grain protein content (GPC) in modern wheat is inherently low. Wild emmer wheat (Triticum turgidum ssp. dicoccoides, 2n = 4x = 28, AABB) gene pool harbors wide genotypic variations in GPC. However, the characterization of candidate genes associated with high GPC is a challenge due to the complex characteristic of this trait. In the current study, we performed RNA-seq analysis on developing grains of wild emmer genotype D1, common wheat CN16, and their hexaploid wide hybrid BAd107-4 with contrasting GPC. We have found a total of 39,795 expressed genes on chromosomes A and B, of which 24,152 were shared between D1, CN16, and BAd107-4. From 1744 differentially expressed genes (DEGs), 1203 were downregulated and 541 were upregulated in the high GPC (D1+BAd107-4) compared with low GPC (CN16) groups. The majority of DEGs were associated with protein processing in endoplasmic reticulum, starch and sucrose metabolism, galactose metabolism, and protein export pathways. Expression levels of nine randomly selected genes were verified by qRT-PCR, which was consistent with the transcriptome data. The present database will help us to understand the potential regulation networks underlying wheat grain protein accumulation and provide the foundation for simultaneous improvement of grain protein content and yield in wheat breeding programs.

Enriching LMW-GS alleles and strengthening gluten properties of common wheat through wide hybridization with wild emmer.

Two advanced lines (BAd7-209 and BAd7-213) with identical high-molecular-weight glutenin subunit composition were obtained via wide hybridization between low-gluten cultivar chuannong16 (CN16) and wild emmer D97 (D97). BAd7-209 was better than BAd7-213, and both of them were much better than CN16 in a dough quality test. We found that BAd7-209 had more abundant and higher expression levels of low-molecular-weight glutenin subunit (LMW-GS) proteins than those of BAd7-213. Twenty-nine novel LMW-GS genes at Glu-A3 locus were isolated from BAd7-209, BAd7-213 and their parents. We found that all 29 LMW-GS genes possessed the same primary structure shared by other known LMW-GSs. Twenty-seven genes encode LMW-m-type subunits, and two encode LMW-i-type subunits. BAd7-209 had a higher number of LMW-GS genes than BAd7-213, CN16, and D97. Two wild emmer genes MG574329 and MG574330 were present in the two advanced lines. Most of the LMW-m-type genes showed minor nucleotide variations between wide hybrids and their parents that could be induced through the wide hybridization process. Our results demonstrated that the wild emmer LMW-GS alleles could be feasibly transferred and integrated into common wheat background via wide hybridization and the potential value of the wild emmer LMW-GS alleles in breeding programs designed to improve wheat flour quality.

Characterization of novel LMW glutenin subunit genes at the Glu-M3 locus from Aegilops comosa.

We report the characterization of seven novel low-molecular-weight glutenin subunit (LMW-GS) genes from Aegilops comosa (2n = 2x = 14, MM). We found that all seven LMW-GS genes possessed the same primary structure shared by other known LMW-GSs. Three genes (comosa-M1, comosa-M2, and comosa-M3) encode LMW-m-type subunits, two (comosa-I1 and comosa-I2) encode LWM-i-type subunits, and two (comosa-L1 and comosa-L2) encode LWM-l-type subunits. The comosa-M1 possessed seven cysteine residues, which resulted from a single-nucleotide polymorphism (SNP) of the G-A transition in the fifth position of the N-terminal sequence. Two l-type subunits, comosa-L1 and comosa-L2, contained nine cysteine residues with an extra cysteine residue located in the signal peptide and repetitive domain. In addition, a long insertion of 13 residues (LGQQPQ8/LVQQPQ8) was present in the end of the C-terminal II. Phylogenetic analysis implied that the comosa-L2 may be the intermediate type during the evolution of LMW-l and LMW-i-type genes. Our results demonstrated that the novel LMW-GSs, such as comosa-M1, comosa-L1, and comosa-L2, may have positive effects on dough properties.