Nitrogen deficiency tolerance conferred by introgression of a QTL derived from wild emmer into bread wheat.

KEY MESSAGE: Genetic dissection of a QTL from wild emmer wheat, QGpc.huj.uh-5B.2, introgressed into bread wheat, identified candidate genes associated with tolerance to nitrogen deficiency, and potentially useful for improving nitrogen-use efficiency. Nitrogen (N) is an important macronutrient critical to wheat growth and development; its deficiency is one of the main factors causing reductions in grain yield and quality. N availability is significantly affected by drought or flooding, that are dependent on additional factors including soil type or duration and severity of stress. In a previous study, we identified a high grain protein content QTL (QGpc.huj.uh-5B.2) derived from the 5B chromosome of wild emmer wheat, that showed a higher proportion of explained variation under water-stress conditions. We hypothesized that this QTL is associated with tolerance to N deficiency as a possible mechanism underlying the higher effect under stress. To validate this hypothesis, we introgressed the QTL into the elite bread wheat var. Ruta, and showed that under N-deficient field conditions the introgression IL99 had a 33% increase in GPC (p < 0.05) compared to the recipient parent. Furthermore, evaluation of IL99 response to severe N deficiency (10% N) for 14 days, applied using a semi-hydroponic system under controlled conditions, confirmed its tolerance to N deficiency. Fine-mapping of the QTL resulted in 26 homozygous near-isogenic lines (BC4F5) segregating to N-deficiency tolerance. The QTL was delimited from - 28.28 to - 1.29 Mb and included 13 candidate genes, most associated with N-stress response, N transport, and abiotic stress responses. These genes may improve N-use efficiency under severely N-deficient environments. Our study demonstrates the importance of WEW as a source of novel candidate genes for sustainable improvement in tolerance to N deficiency in wheat.

Genome sequences of three Aegilops species of the section Sitopsis reveal phylogenetic relationships and provide resources for wheat improvement.

Aegilops is a close relative of wheat (Triticum spp.), and Aegilops species in the section Sitopsis represent a rich reservoir of genetic diversity for the improvement of wheat. To understand their diversity and advance their utilization, we produced whole-genome assemblies of Aegilops longissima and Aegilops speltoides. Whole-genome comparative analysis, along with the recently sequenced Aegilops sharonensis genome, showed that the Ae. longissima and Ae. sharonensis genomes are highly similar and are most closely related to the wheat D subgenome. By contrast, the Ae. speltoides genome is more closely related to the B subgenome. Haplotype block analysis supported the idea that Ae. speltoides genome is closest to the wheat B subgenome, and highlighted variable and similar genomic regions between the three Aegilops species and wheat. Genome-wide analysis of nucleotide-binding leucine-rich repeat (NLR) genes revealed species-specific and lineage-specific NLR genes and variants, demonstrating the potential of Aegilops genomes for wheat improvement.

Wild emmer introgression alters root-to-shoot growth dynamics in durum wheat in response to water stress.

Water deficit during the early vegetative growth stages of wheat (Triticum) can limit shoot growth and ultimately impact grain productivity. Introducing diversity in wheat cultivars to enhance the range of phenotypic responses to water limitations during vegetative growth can provide potential avenues for mitigating subsequent yield losses. We tested this hypothesis in an elite durum wheat background by introducing a series of introgressions from a wild emmer (Triticum turgidum ssp. dicoccoides) wheat. Wild emmer populations harbor rich phenotypic diversity for drought-adaptive traits. To determine the effect of these introgressions on vegetative growth under water-limited conditions, we used image-based phenotyping to catalog divergent growth responses to water stress ranging from high plasticity to high stability. One of the introgression lines exhibited a significant shift in root-to-shoot ratio in response to water stress. We characterized this shift by combining genetic analysis and root transcriptome profiling to identify candidate genes (including a root-specific kinase) that may be linked to the root-to-shoot carbon reallocation under water stress. Our results highlight the potential of introducing functional diversity into elite durum wheat for enhancing the range of water stress adaptation.

Genome-Wide Mapping of Loci for Adult-Plant Resistance to Stripe Rust in Durum Wheat Svevo Using the 90K SNP Array.

Stripe rust is a foliar disease in wheat caused by Puccinia striiformis f. tritici. The best way to protect wheat from this disease is by growing resistant cultivars. Tetraploid wheat can serve as a good source of valuable genetic diversity for various traits. Here, we report the mapping of nine stripe rust resistance quantitative trait loci (QTL) effective against P. striiformis f. tritici in China and Israel. We used recombinant inbred lines (RILs) developed from a cross between the durum wheat cultivar Svevo and Triticum dicoccoides accession Zavitan. By genotyping the RIL population of 137 lines using the wheat 90K single-nucleotide polymorphism array, we mapped an adult-plant resistance locus QYrsv.swust-1BL.1, the most effective QTL, within a 0.75-centimorgan region in T. turgidum subsp. durum 'Svevo' on chromosome arm 1BL, corresponding to the region of 670.7 to 671.5 Mb on the Chinese Spring chromosome arm 1BL. Of the other eight minor-effect stripe rust QTL, seven were from Svevo and mapped on chromosomes 1A, 1B, 2B, 3A, 4A, and 5A, and one was from Zavitan and mapped on chromosome 2A. Several QTL with epistatic effects were identified as well. The markers linked to the resistance QTL can be useful in marker-assisted selection for incorporation of these resistance QTL into both durum and common wheat cultivars.

Structural variation and rates of genome evolution in the grass family seen through comparison of sequences of genomes greatly differing in size.

Homology was searched with genes annotated in the Aegilops tauschii pseudomolecules against genes annotated in the pseudomolecules of tetraploid wild emmer wheat, Brachypodium distachyon, sorghum and rice. Similar searches were performed with genes annotated in the rice pseudomolecules. Matrices of collinear genes and rearrangements in their order were constructed. Optical BioNano genome maps were constructed and used to validate rearrangements unique to the wild emmer and Ae. tauschii genomes. Most common rearrangements were short paracentric inversions and short intrachromosomal translocations. Intrachromosomal translocations outnumbered segmental intrachromosomal duplications. The densities of paracentric inversion lengths were approximated by exponential distributions in all six genomes. Densities of collinear genes along the Ae. tauschii chromosomes were highly correlated with meiotic recombination rates but those of rearrangements were not, suggesting different causes of the erosion of gene collinearity and evolution of major chromosome rearrangements. Frequent rearrangements sharing breakpoints suggested that chromosomes have been rearranged recurrently at some sites. The distal 4 Mb of the short arms of rice chromosomes Os11 and Os12 and corresponding regions in the sorghum, B. distachyon and Triticeae genomes contain clusters of interstitial translocations including from 1 to 7 collinear genes. The rates of acquisition of major rearrangements were greater in the large wild emmer wheat and Ae. tauschii genomes than in the lineage preceding their divergence or in the B. distachyon, rice and sorghum lineages. It is suggested that synergy between large quantities of dynamic transposable elements and annual growth habit have been the primary causes of the fast evolution of the Triticeae genomes.

Rapid evolution of α-gliadin gene family revealed by analyzing Gli-2 locus regions of wild emmer wheat.

α-Gliadins are a major group of gluten proteins in wheat flour that contribute to the end-use properties for food processing and contain major immunogenic epitopes that can cause serious health-related issues including celiac disease (CD). α-Gliadins are also the youngest group of gluten proteins and are encoded by a large gene family. The majority of the gene family members evolved independently in the A, B, and D genomes of different wheat species after their separation from a common ancestral species. To gain insights into the origin and evolution of these complex genes, the genomic regions of the Gli-2 loci encoding α-gliadins were characterized from the tetraploid wild emmer, a progenitor of hexaploid bread wheat that contributed the AABB genomes. Genomic sequences of Gli-2 locus regions for the wild emmer A and B genomes were first reconstructed using the genome sequence scaffolds along with optical genome maps. A total of 24 and 16 α-gliadin genes were identified for the A and B genome regions, respectively. α-Gliadin pseudogene frequencies of 86% for the A genome and 69% for the B genome were primarily caused by C to T substitutions in the highly abundant glutamine codons, resulting in the generation of premature stop codons. Comparison with the homologous regions from the hexaploid wheat cv. Chinese Spring indicated considerable sequence divergence of the two A genomes at the genomic level. In comparison, conserved regions between the two B genomes were identified that included α-gliadin pseudogenes containing shared nested TE insertions. Analyses of the genomic organization and phylogenetic tree reconstruction indicate that although orthologous gene pairs derived from speciation were present, large portions of α-gliadin genes were likely derived from differential gene duplications or deletions after the separation of the homologous wheat genomes ~ 0.5 MYA. The higher number of full-length intact α-gliadin genes in hexaploid wheat than that in wild emmer suggests that human selection through domestication might have an impact on α-gliadin evolution. Our study provides insights into the rapid and dynamic evolution of genomic regions harboring the α-gliadin genes in wheat.

A Metabolic Gene Cluster in the Wheat W1 and the Barley Cer-cqu Loci Determines ß-Diketone Biosynthesis and Glaucousness.

The glaucous appearance of wheat (Triticum aestivum) and barley (Hordeum vulgare) plants, that is the light bluish-gray look of flag leaf, stem, and spike surfaces, results from deposition of cuticular ß-diketone wax on their surfaces; this phenotype is associated with high yield, especially under drought conditions. Despite extensive genetic and biochemical characterization, the molecular genetic basis underlying the biosynthesis of ß-diketones remains unclear. Here, we discovered that the wheat W1 locus contains a metabolic gene cluster mediating ß-diketone biosynthesis. The cluster comprises genes encoding proteins of several families including type-III polyketide synthases, hydrolases, and cytochrome P450s related to known fatty acid hydroxylases. The cluster region was identified in both genetic and physical maps of glaucous and glossy tetraploid wheat, demonstrating entirely different haplotypes in these accessions. Complementary evidence obtained through gene silencing in planta and heterologous expression in bacteria supports a model for a ß-diketone biosynthesis pathway involving members of these three protein families. Mutations in homologous genes were identified in the barley eceriferum mutants defective in ß-diketone biosynthesis, demonstrating a gene cluster also in the ß-diketone biosynthesis Cer-cqu locus in barley. Hence, our findings open new opportunities to breed major cereal crops for surface features that impact yield and stress response.

GNI-A1 mediates trade-off between grain number and grain weight in tetraploid wheat.

KEY MESSAGE: Wild emmer allele of GNI-A1 ease competition among developing grains through the suppression of floret fertility and increase grain weight in tetraploid wheat. Grain yield is a highly polygenic trait determined by the number of grains per unit area, as well as by grain weight. In wheat, grain number and grain weight are usually negatively correlated. Yet, the genetic basis underlying trade-off between the two is mostly unknown. Here, we fine-mapped a grain weight QTL using wild emmer introgressions in a durum wheat background and showed that grain weight is associated with the GNI-A1 gene, a regulator of floret fertility. In-depth characterization of grain number and grain weight indicated that suppression of distal florets by the wild emmer GNI-A1 allele increases weight of proximal grains in basal and central spikelets due to alteration in assimilate distribution. Re-sequencing of GNI-A1 in tetraploid wheat demonstrated the rich allelic repertoire of the wild emmer gene pool, including a rare allele which was present in two gene copies and contained a nonsynonymous mutation in the C-terminus of the protein. Using an F2 population generated from a cross between wild emmer accessions Zavitan, which carries the rare allele, and TTD140, we demonstrated that this unique polymorphism is associated with grain weight, independent of grain number. Moreover, we showed, for the first time, that GNI-A1 proteins are transcriptional activators and that selection targeted compromised activity of the protein. Our findings expand the knowledge of the genetic basis underlying trade-off between key yield components and may contribute to breeding efforts for enhanced grain yield.

Recombination between homoeologous chromosomes induced in durum wheat by the Aegilops speltoides Su1-Ph1 suppressor.

KEY MESSAGE: Su1-Ph1, which we previously introgressed into wheat from Aegilops speltoides, is a potent suppressor of Ph1 and a valuable tool for gene introgression in tetraploid wheat. We previously introgressed Su1-Ph1, a suppressor of the wheat Ph1 gene, from Aegilops speltoides into durum wheat cv Langdon (LDN). Here, we evaluated the utility of the introgressed suppressor for inducing introgression of alien germplasm into durum wheat. We built LDN plants heterozygous for Su1-Ph1 that simultaneously contained a single LDN chromosome 5B and a single Ae. searsii chromosome 5Sse, which targeted them for recombination. We genotyped 28 BC1F1 and 84 F2 progeny with the wheat 90-K Illumina single-nucleotide polymorphism assay and detected extensive recombination between the two chromosomes, which we confirmed by non-denaturing fluorescence in situ hybridization (ND-FISH). We constructed BC1F1 and F2 genetic maps that were 65.31 and 63.71 cM long, respectively. Recombination rates between the 5B and 5Sse chromosomes were double the expected rate computed from their meiotic pairing, which we attributed to selection against aneuploid gametes. Recombination rate between 5B and 5Sse was depressed compared to that between 5B chromosomes in the proximal region of the long arm. We integrated ND-FISH signals into the genetic map and constructed a physical map, which we used to map a 172,188,453-bp Ph1 region. Despite the location of the region in a low-recombination region of the 5B chromosome, we detected three crossovers in it. Our data show that Su1-Ph1 is a valuable tool for gene introgression and gene mapping based on recombination between homoeologous chromosomes in wheat.

Correction to: Recombination between homoeologous chromosomes induced in durum wheat by the Aegilops speltoides Su1-Ph1 suppressor.

Unfortunately, the 9th author name was incorrectly published in the original publication. The complete correct name is given below.

Chromosome-based survey sequencing reveals the genome organization of wild wheat progenitor Triticum dicoccoides.

Wild emmer wheat (Triticum turgidum ssp. dicoccoides) is the progenitor of wheat. We performed chromosome-based survey sequencing of the 14 chromosomes, examining repetitive sequences, protein-coding genes, miRNA/target pairs and tRNA genes, as well as syntenic relationships with related grasses. We found considerable differences in the content and distribution of repetitive sequences between the A and B subgenomes. The gene contents of individual chromosomes varied widely, not necessarily correlating with chromosome size. We catalogued candidate agronomically important loci, along with new alleles and flanking sequences that can be used to design exome sequencing. Syntenic relationships and virtual gene orders revealed several small-scale evolutionary rearrangements, in addition to providing evidence for the 4AL-5AL-7BS translocation in wild emmer wheat. Chromosome-based sequence assemblies contained five novel miRNA families, among 59 families putatively encoded in the entire genome which provide insight into the domestication of wheat and an overview of the genome content and organization.

A glycosyl transferase family 43 protein involved in xylan biosynthesis is associated with straw digestibility in Brachypodium distachyon.

The recalcitrance of secondary plant cell walls to digestion constrains biomass use for the production of sustainable bioproducts and for animal feed. We screened a population of Brachypodium recombinant inbred lines (RILs) for cell wall digestibility using commercial cellulases and detected a quantitative trait locus (QTL) associated with this trait. Examination of the chromosomal region associated with this QTL revealed a candidate gene that encodes a putative glycosyl transferase family (GT) 43 protein, orthologue of IRX14 in Arabidopsis, and hence predicted to be involved in the biosynthesis of xylan. Arabinoxylans form the major matrix polysaccharides in cell walls of grasses, such as Brachypodium. The parental lines of the RIL population carry alternative nonsynonymous polymorphisms in the BdGT43A gene, which were inherited in the RIL progeny in a manner compatible with a causative role in the variation in straw digestibility. In order to validate the implied role of our candidate gene in affecting straw digestibility, we used RNA interference to lower the expression levels of the BdGT43A gene in Brachypodium. The biomass of the silenced lines showed higher digestibility supporting a causative role of the BdGT43A gene, suggesting that it might form a good target for improving straw digestibility in crops.

SNP-based pool genotyping and haplotype analysis accelerate fine-mapping of the wheat genomic region containing stripe rust resistance gene Yr26.

KEY MESSAGE: NGS-assisted super pooling emerging as powerful tool to accelerate gene mapping and haplotype association analysis within target region uncovering specific linkage SNPs or alleles for marker-assisted gene pyramiding. Conventional gene mapping methods to identify genes associated with important agronomic traits require significant amounts of financial support and time. Here, a single nucleotide polymorphism (SNP)-based mapping approach, RNA-Seq and SNP array assisted super pooling analysis, was used for rapid mining of a candidate genomic region for stripe rust resistance gene Yr26 that has been widely used in wheat breeding programs in China. Large DNA and RNA super-pools were genotyped by Wheat SNP Array and sequenced by Illumina HiSeq, respectively. Hundreds of thousands of SNPs were identified and then filtered by multiple filtering criteria. Among selected SNPs, over 900 were found within an overlapping interval of less than 30 Mb as the Yr26 candidate genomic region in the centromeric region of chromosome arm 1BL. The 235 chromosome-specific SNPs were converted into KASP assays to validate the Yr26 interval in different genetic populations. Using a high-resolution mapping population (> 30,000 gametes), we confined Yr26 to a 0.003-cM interval. The Yr26 target region was anchored to the common wheat IWGSC RefSeq v1.0 and wild emmer WEWSeq v.1.0 sequences, from which 488 and 454 kb fragments were obtained. Several candidate genes were identified in the target genomic region, but there was no typical resistance gene in either genome region. Haplotype analysis identified specific SNPs linked to Yr26 and developed robust and breeder-friendly KASP markers. This integration strategy can be applied to accelerate generating many markers closely linked to target genes/QTL for a trait of interest in wheat and other polyploid species.

Reassessment of the evolution of wheat chromosomes 4A, 5A, and 7B.

KEY MESSAGE: Comparison of genome sequences of wild emmer wheat and Aegilops tauschii suggests a novel scenario of the evolution of rearranged wheat chromosomes 4A, 5A, and 7B. Past research suggested that wheat chromosome 4A was subjected to a reciprocal translocation T(4AL;5AL)1 that occurred in the diploid progenitor of the wheat A subgenome and to three major rearrangements that occurred in polyploid wheat: pericentric inversion Inv(4AS;4AL)1, paracentric inversion Inv(4AL;4AL)1, and reciprocal translocation T(4AL;7BS)1. Gene collinearity along the pseudomolecules of tetraploid wild emmer wheat (Triticum turgidum ssp. dicoccoides, subgenomes AABB) and diploid Aegilops tauschii (genomes DD) was employed to confirm these rearrangements and to analyze the breakpoints. The exchange of distal regions of chromosome arms 4AS and 4AL due to pericentric inversion Inv(4AS;4AL)1 was detected, and breakpoints were validated with an optical Bionano genome map. Both breakpoints contained satellite DNA. The breakpoints of reciprocal translocation T(4AL;7BS)1 were also found. However, the breakpoints that generated paracentric inversion Inv(4AL;4AL)1 appeared to be collocated with the 4AL breakpoints that had produced Inv(4AS;4AL)1 and T(4AL;7BS)1. Inv(4AS;4AL)1, Inv(4AL;4AL)1, and T(4AL;7BS)1 either originated sequentially, and Inv(4AL;4AL)1 was produced by recurrent chromosome breaks at the same breakpoints that generated Inv(4AS;4AL)1 and T(4AL;7BS)1, or Inv(4AS;4AL)1, Inv(4AL;4AL)1, and T(4AL;7BS)1 originated simultaneously. We prefer the latter hypothesis since it makes fewer assumptions about the sequence of events that produced these chromosome rearrangements.

Acceleration of leaf senescence is slowed down in transgenic barley plants deficient in the DNA/RNA-binding protein WHIRLY1.

WHIRLY1 in barley was isolated as a potential regulator of the senescence-associated gene HvS40. In order to investigate whether the plastid-nucleus-located DNA/RNA-binding protein WHIRLY1 plays a role in regulation of leaf senescence, primary foliage leaves from transgenic barley plants with an RNAi-mediated knockdown of the WHIRLY1 gene were characterized by typical senescence parameters, namely pigment contents, function and composition of the photosynthetic apparatus, as well as expression of selected genes known to be either down- or up-regulated during leaf senescence. When the plants were grown at low light intensity, senescence progression was similar between wild-type and RNAi-W1 plants. Likewise, dark-induced senescence of detached leaves was not affected by reduction of WHIRLY1. When plants were grown at high light intensity, however, senescence was induced prematurely in wild-type plants but was delayed in RNAi-W1 plants. This result suggests that WHIRLY1 plays a role in light sensing and/or stress communication between chloroplasts and the nucleus.

The Solanum tuberosum KST1 partial promoter as a tool for guard cell expression in multiple plant species.

To date, guard cell promoters have been examined in only a few species, primarily annual dicots. A partial segment of the potato (Solanum tuberosum) KST1 promoter (KST1 partial promoter, KST1ppro) has previously been shown to confer guard cell expression in potato, tomato (Solanum lycopersicum), citrus [Troyer citrange (C. sinensis×Poncirus trifoliata)], and Arabidopsis (Arabidopsis thaliana). Here, we describe an extensive analysis of the expression pattern of KST1ppro in eight (previously reported, as well as new) species from five different angiosperm families, including the Solanaceae and the Cucurbitaceae, Arabidopsis, the monocot barley (Hordeum vulgare), and two perennial species: grapevine (Vitis vinifera) and citrus. Using confocal imaging and three-dimensional movies, we demonstrate that KST1ppro drives guard cell expression in all of these species, making it the first dicot-originated guard cell promoter shown to be active in a monocot and the first promoter reported to confer guard cell expression in barley and cucumber (Cucumis sativus). The results presented here indicate that KST1ppro can be used to drive constitutive guard cell expression in monocots and dicots and in both annual and perennial plants. In addition, we show that the KST1ppro is active in guard cells shortly after the symmetric division of the guard mother cell and generates stable expression in mature guard cells. This allows us to follow the spatial and temporal distribution of stomata in cotyledons and true leaves.

QTLs for uniform grain dimensions and germination selected during wheat domestication are co-located on chromosome 4B.

KEY MESSAGE: A major locus on the long arm of wheat chromosome 4B controls within-spikelet variation in both grain size and seed dormancy, the latter an important survival mechanism likely eliminated from wild wheat during domestication. Seed dormancy can increase the probability of survival of at least some progeny under unstable environmental conditions. In wild emmer wheat, only one of the two grains in a spikelet germinates during the first rainy season following maturation; and this within-plant variation in seed dormancy is associated with both grain dimension differences and position within the spikelet. Here, in addition to characterizing these associations, we elucidate the genetic mechanism controlling differential grain dimensions and dormancy within wild tetraploid wheat spikelets using phenotypic data from a wild emmer × durum wheat population and a high-density genetic map. We show that in wild emmer, the lower grain within the spikelet is about 30 % smaller and more dormant than the larger, upper grain that germinates usually within 3 days. We identify a major locus on the long arm of chromosome 4B that explains >40 % of the observed variation in grain dimensions and seed dormancy within spikelets. This locus, designated QGD-4BL, is validated using an independent set of wild emmer × durum wheat genetic stocks. The domesticated variant of this novel locus on chromosome 4B, likely fixed during the process of wheat domestication, favors spikelets with seeds of uniform size and synchronous germination. The identification of locus QGD-4BL enhances our knowledge of the genetic basis of the domestication syndrome of one of our most important crops.

A high-density, SNP-based consensus map of tetraploid wheat as a bridge to integrate durum and bread wheat genomics and breeding.

Consensus linkage maps are important tools in crop genomics. We have assembled a high-density tetraploid wheat consensus map by integrating 13 data sets from independent biparental populations involving durum wheat cultivars (Triticum turgidum ssp. durum), cultivated emmer (T. turgidum ssp. dicoccum) and their ancestor (wild emmer, T. turgidum ssp. dicoccoides). The consensus map harboured 30 144 markers (including 26 626 SNPs and 791 SSRs) half of which were present in at least two component maps. The final map spanned 2631 cM of all 14 durum wheat chromosomes and, differently from the individual component maps, all markers fell within the 14 linkage groups. Marker density per genetic distance unit peaked at centromeric regions, likely due to a combination of low recombination rate in the centromeric regions and even gene distribution along the chromosomes. Comparisons with bread wheat indicated fewer regions with recombination suppression, making this consensus map valuable for mapping in the A and B genomes of both durum and bread wheat. Sequence similarity analysis allowed us to relate mapped gene-derived SNPs to chromosome-specific transcripts. Dense patterns of homeologous relationships have been established between the A- and B-genome maps and between nonsyntenic homeologous chromosome regions as well, the latter tracing to ancient translocation events. The gene-based homeologous relationships are valuable to infer the map location of homeologs of target loci/QTLs. Because most SNP and SSR markers were previously mapped in bread wheat, this consensus map will facilitate a more effective integration and exploitation of genes and QTL for wheat breeding purposes.

Regulation of Zn and Fe transporters by the GPC1 gene during early wheat monocarpic senescence.

BACKGROUND: During wheat senescence, leaf components are degraded in a coordinated manner, releasing amino acids and micronutrients which are subsequently transported to the developing grain. We have previously shown that the simultaneous downregulation of Grain Protein Content (GPC) transcription factors, GPC1 and GPC2, greatly delays senescence and disrupts nutrient remobilization, and therefore provide a valuable entry point to identify genes involved in micronutrient transport to the wheat grain. RESULTS: We generated loss-of-function mutations for GPC1 and GPC2 in tetraploid wheat and showed in field trials that gpc1 mutants exhibit significant delays in senescence and reductions in grain Zn and Fe content, but that mutations in GPC2 had no significant effect on these traits. An RNA-seq study of these mutants at different time points showed a larger proportion of senescence-regulated genes among the GPC1 (64%) than among the GPC2 (37%) regulated genes. Combined, the two GPC genes regulate a subset (21.2%) of the senescence-regulated genes, 76.1% of which are upregulated at 12 days after anthesis, before the appearance of any visible signs of senescence. Taken together, these results demonstrate that GPC1 is a key regulator of nutrient remobilization which acts predominantly during the early stages of senescence. Genes upregulated at this stage include transporters from the ZIP and YSL gene families, which facilitate Zn and Fe export from the cytoplasm to the phloem, and genes involved in the biosynthesis of chelators that facilitate the phloem-based transport of these nutrients to the grains. CONCLUSIONS: This study provides an overview of the transport mechanisms activated in the wheat flag leaf during monocarpic senescence. It also identifies promising targets to improve nutrient remobilization to the wheat grain, which can help mitigate Zn and Fe deficiencies that afflict many regions of the developing world.

Functional characterization of GPC-1 genes in hexaploid wheat.

In wheat, monocarpic senescence is a tightly regulated process during which nitrogen (N) and micronutrients stored pre-anthesis are remobilized from vegetative tissues to the developing grains. Recently, a close connection between senescence and remobilization was shown through the map-based cloning of the GPC (grain protein content) gene in wheat. GPC-B1 encodes a NAC transcription factor associated with earlier senescence and increased grain protein, iron and zinc content, and is deleted or non-functional in most commercial wheat varieties. In the current research, we identified 'loss of function' ethyl methanesulfonate mutants for the two GPC-B1 homoeologous genes; GPC-A1 and GPC-D1, in a hexaploid wheat mutant population. The single gpc-a1 and gpc-d1 mutants, the double gpc-1 mutant and control lines were grown under field conditions at four locations and were characterized for senescence, GPC, micronutrients and yield parameters. Our results show a significant delay in senescence in both the gpc-a1 and gpc-d1 single mutants and an even stronger effect in the gpc-1 double mutant in all the environments tested in this study. The accumulation of total N in the developing grains showed a similar increase in the control and gpc-1 plants until 25 days after anthesis (DAA) but at 41 and 60 DAA the control plants had higher grain N content than the gpc-1 mutants. At maturity, GPC in all mutants was significantly lower than in control plants while grain weight was unaffected. These results demonstrate that the GPC-A1 and GPC-D1 genes have a redundant function and play a major role in the regulation of monocarpic senescence and nutrient remobilization in wheat.