Multiple wheat genomes reveal global variation in modern breeding.

Advances in genomics have expedited the improvement of several agriculturally important crops but similar efforts in wheat (Triticum spp.) have been more challenging. This is largely owing to the size and complexity of the wheat genome1, and the lack of genome-assembly data for multiple wheat lines2,3. Here we generated ten chromosome pseudomolecule and five scaffold assemblies of hexaploid wheat to explore the genomic diversity among wheat lines from global breeding programs. Comparative analysis revealed extensive structural rearrangements, introgressions from wild relatives and differences in gene content resulting from complex breeding histories aimed at improving adaptation to diverse environments, grain yield and quality, and resistance to stresses4,5. We provide examples outlining the utility of these genomes, including a detailed multi-genome-derived nucleotide-binding leucine-rich repeat protein repertoire involved in disease resistance and the characterization of Sm16, a gene associated with insect resistance. These genome assemblies will provide a basis for functional gene discovery and breeding to deliver the next generation of modern wheat cultivars.

Spatiotemporal transcriptomics and metabolic profiling provide insights into gene regulatory networks during lentil seed development.

Lentil (Lens culinaris Medik.) is a nutritious legume with seeds rich in protein, minerals and an array of diverse specialized metabolites. The formation of a seed requires regulation and tight coordination of developmental programs to form the embryo, endosperm and seed coat compartments, which determines the structure and composition of mature seed and thus its end-use quality. Understanding the molecular and cellular events and metabolic processes of seed development is essential for improving lentil yield and seed nutritional value. However, such information remains largely unknown, especially at the seed compartment level. In this study, we generated high-resolution spatiotemporal gene expression profiles in lentil embryo, seed coat and whole seeds from fertilization through maturation. Apart from anatomic differences between the embryo and seed coat, comparative transcriptomics and weighted gene co-expression network analysis revealed embryo- and seed coat-specific genes and gene modules predominant in specific tissues and stages, which highlights distinct genetic programming. Furthermore, we investigated the dynamic profiles of flavonoid, isoflavone, phytic acid and saponin in seed compartments across seed development. Coupled with transcriptome data, we identified sets of candidate genes involved in the biosynthesis of these metabolites. The global view of the transcriptional and metabolic changes of lentil seed tissues throughout development provides a valuable resource for dissecting the genetic control of secondary metabolism and development of molecular tools for improving seed nutritional quality.

Systematic Characterization of Multi-Rust Resistance Genes from a 'Parula × Thatcher' Population with a High-Density Genetic Map.

Pyramiding multiple resistant genes has been proposed as the most effective way to control wheat rust diseases globally. Identifying the most effective pyramids is challenged by the large pool of rust resistance genes and limited information about their mechanisms of resistance and interactions. Here, using a high-density genetic map, a double haploid population, and multi-rust field testing, we aimed to systematically characterize the most effective gene pyramids for rust resistance from the durable multi-rust resistant CIMMYT cultivar Parula. We revealed that the Parula resistance gene pyramid contains Lr34/Yr18/Sr57 (Lr34), Lr46/Yr29/Sr58 (Lr46), Lr27/Yr30/Sr2 (Sr2), and Lr68. The efficacy, magnitude of effect, and interactions varied for the three rust diseases. A subpopulation mapping approach was applied to characterize the complex interactions of the resistance genes by controlling for the effect of Lr34. Using this approach, we found that Lr34 and Lr68 have a strong additive effect for leaf rust, whereas no additive effects were observed for any rusts between Lr34 and Lr46. Lr34 combined synergistically with Sr12 from Thatcher for stem rust, whereas the additive effect of Lr34 and Sr2 was dependent on the type of rust and environment. Two novel leaf rust quantitative trait loci (QTLs) from Parula were identified in this study, a stable QTL QLr-7BS and QLr-5AS, which showed Lr34 dependent expression. With these findings, we propose combining two to three high-value genes from Canadian wheat (e.g., Sr12 from Thatcher) with a foundational multi-adult plant resistance cassette for desirable and durable resistance to all three rusts in Canadian wheat.

Weighted gene co-expression network analysis unveils gene networks associated with the Fusarium head blight resistance in tetraploid wheat.

BACKGROUND: Fusarium head blight (FHB) resistance in the durum wheat breeding gene pool is rarely reported. Triticum turgidum ssp. carthlicum line Blackbird is a tetraploid relative of durum wheat that offers partial FHB resistance. Resistance QTL were identified for the durum wheat cv. Strongfield × Blackbird population on chromosomes 1A, 2A, 2B, 3A, 6A, 6B and 7B in a previous study. The objective of this study was to identify the defense mechanisms underlying the resistance of Blackbird and report candidate regulator defense genes and single nucleotide polymorphism (SNP) markers within these genes for high-resolution mapping of resistance QTL reported for the durum wheat cv. Strongfield/Blackbird population. RESULTS: Gene network analysis identified five networks significantly (P < 0.05) associated with the resistance to FHB spread (Type II FHB resistance) one of which showed significant correlation with both plant height and relative maturity traits. Two gene networks showed subtle differences between Fusarium graminearum-inoculated and mock-inoculated plants, supporting their involvement in constitutive defense. The candidate regulator genes have been implicated in various layers of plant defense including pathogen recognition (mainly Nucleotide-binding Leucine-rich Repeat proteins), signaling pathways including the abscisic acid and mitogen activated protein (MAP) kinase, and downstream defense genes activation including transcription factors (mostly with dual roles in defense and development), and cell death regulator and cell wall reinforcement genes. The expression of five candidate genes measured by quantitative real-time PCR was correlated with that of RNA-seq, corroborating the technical and analytical accuracy of RNA-sequencing. CONCLUSIONS: Gene network analysis allowed identification of candidate regulator genes and genes associated with constitutive resistance, those that will not be detected using traditional differential expression analysis. This study also shed light on the association of developmental traits with FHB resistance and partially explained the co-localization of FHB resistance with plant height and maturity QTL reported in several previous studies. It also allowed the identification of candidate hub genes within the interval of three previously reported FHB resistance QTL for the Strongfield/Blackbird population and associated SNPs for future high resolution mapping studies.

Arabidopsis UBC13 differentially regulates two programmed cell death pathways in responses to pathogen and low-temperature stress.

UBC13 is required for Lys63-linked polyubiquitination and innate immune responses in mammals, but its functions in plant immunity remain to be defined. Here we used genetic and pathological methods to evaluate roles of Arabidopsis UBC13 in response to pathogens and environmental stresses. Loss of UBC13 failed to activate the expression of numerous cold-responsive genes and resulted in hypersensitivity to low-temperature stress, indicating that UBC13 is involved in plant response to low-temperature stress. Furthermore, the ubc13 mutant displayed low-temperature-induced and salicylic acid-dependent lesion mimic phenotypes. Unlike typical lesion mimic mutants, ubc13 did not enhance disease resistance against virulent bacterial and fungal pathogens, but diminished hypersensitive response and compromised effector-triggered immunity against avirulent bacterial pathogens. UBC13 differently regulates two types of programmed cell death in response to low temperature and pathogen. The lesion mimic phenotype in the ubc13 mutant is partially dependent on SNC1. UBC13 interacts with an F-box protein CPR1 that regulates the homeostasis of SNC1. However, the SNC1 protein level was not altered in the ubc13 mutant, implying that UBC13 is not involved in CPR1-regulated SNC1 protein degradation. Taken together, our results revealed that UBC13 is a key regulator in plant response to low temperature and pathogens.

Highly predictive SNP markers for efficient selection of the wheat leaf rust resistance gene Lr16.

BACKGROUND: Lr16 is a widely deployed leaf rust resistance gene in wheat (Triticum aestivum L.) that is highly effective against the North American Puccinia triticina population when pyramided with the gene Lr34. Lr16 is a seedling leaf rust resistance gene conditioning an incompatible interaction with a distinct necrotic ring surrounding the uredinium. Lr16 was previously mapped to the telomeric region of the short arm of wheat chromosome 2B. The goals of this study were to develop numerous single nucleotide polymorphism (SNP) markers for the Lr16 region and identify diagnostic gene-specific SNP marker assays for marker-assisted selection (MAS). RESULTS: Forty-three SNP markers were developed and mapped on chromosome 2BS tightly linked with the resistance gene Lr16 across four mapping populations representing a total of 1528 gametes. Kompetitive Allele Specific PCR (KASP) assays were designed for all identified SNPs. Resistance gene analogs (RGAs) linked with the Lr16 locus were identified and RGA-based SNP markers were developed. The diagnostic potential of the SNPs co-segregating with Lr16 was evaluated in a diverse set of 133 cultivars and breeding lines. Six SNP markers were consistent with the Lr16 phenotype and are accurately predictive of Lr16 for all wheat lines/cultivars in the panel. CONCLUSIONS: Lr16 was mapped relative to SNP markers in four populations. Six SNP markers exhibited high quality clustering in the KASP assay and are suitable for MAS of Lr16 in wheat breeding programs.

A Systems Genetics Approach Identifies Gene Regulatory Networks Associated with Fatty Acid Composition in Brassica rapa Seed.

Fatty acids in seeds affect seed germination and seedling vigor, and fatty acid composition determines the quality of seed oil. In this study, quantitative trait locus (QTL) mapping of fatty acid and transcript abundance was integrated with gene network analysis to unravel the genetic regulation of seed fatty acid composition in a Brassica rapa doubled haploid population from a cross between a yellow sarson oil type and a black-seeded pak choi. The distribution of major QTLs for fatty acids showed a relationship with the fatty acid types: linkage group A03 for monounsaturated fatty acids, A04 for saturated fatty acids, and A05 for polyunsaturated fatty acids. Using a genetical genomics approach, expression quantitative trait locus (eQTL) hotspots were found at major fatty acid QTLs on linkage groups A03, A04, A05, and A09. An eQTL-guided gene coexpression network of lipid metabolism-related genes showed major hubs at the genes BrPLA2-ALPHA, BrWD-40, a number of seed storage protein genes, and the transcription factor BrMD-2, suggesting essential roles for these genes in lipid metabolism. Three subnetworks were extracted for the economically important and most abundant fatty acids erucic, oleic, linoleic, and linolenic acids. Network analysis, combined with comparison of the genome positions of cis- or trans-eQTLs with fatty acid QTLs, allowed the identification of candidate genes for genetic regulation of these fatty acids. The generated insights in the genetic architecture of fatty acid composition and the underlying complex gene regulatory networks in B. rapa seeds are discussed.

Genetics and mapping of seedling resistance to Ug99 stem rust in winter wheat cultivar Triumph 64 and differentiation of SrTmp, SrCad, and Sr42.

KEY MESSAGE: Resistance to Ug99 stem rust in Triumph 64 was conferred by SrTmp on chromosome arm 6DS and was mapped to the same position as SrCad and Sr42 , however, the three genes show functional differences. Stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is an important disease of wheat that can be controlled by effective stem rust resistance (Sr) genes. The emergence of virulent Pgt races in Africa, namely Ug99 and its variants, has stimulated the search for new Sr genes and genetic characterization of known sources of resistance. Triumph 64 is a winter wheat cultivar that carries gene SrTmp, which confers resistance to Ug99. The goals of this study were to genetically map SrTmp and examine its relationship with other Sr genes occupying a similar chromosome location. A doubled haploid (DH) population from the cross LMPG-6S/Triumph 64 was inoculated with Ug99 at the seedling stage. A single gene conditioning resistance to Ug99 segregated in the population. Genetic mapping with SSR markers placed SrTmp on chromosome arm 6DS in a region similar to SrCad and Sr42. SNP markers developed for SrCad were used to further map SrTmp and were also added to a genetic map of Sr42 using a DH population (LMPG-6S/Norin 40). Three SNP markers that co-segregated with SrTmp also co-segregated with SrCad and Sr42. The SNP markers showed no difference in the map locations of SrTmp, SrCad, and Sr42. Multi-race testing with DH lines from the Triumph 64 and Norin 40 populations and a recombinant inbred-line population from the cross LMPG-6S/AC Cadillac showed that SrTmp, SrCad, and Sr42 confer different spectra of resistance. Markers closely linked to SrTmp are suitable for marker-assisted breeding and germplasm development.

Genetic mapping of SrCad and SNP marker development for marker-assisted selection of Ug99 stem rust resistance in wheat.

KEY MESSAGE: New SNP markers that can be used for marker-assisted selection and map-based cloning saturate the chromosome region carrying SrCad , a wheat gene that confers resistance to Ug99 stem rust. Wheat stem rust, caused by Puccinia graminis f. sp. tritici, is a devastating disease of wheat worldwide. Development of cultivars with effective resistance has been the primary means to control this disease, but the appearance of new virulent strains such as Ug99 has rendered most wheat varieties vulnerable. The stem rust resistance gene SrCad located on chromosome arm 6DS has provided excellent resistance to various strains of Ug99 in field nurseries conducted in Njoro, Kenya since 2005. Three genetic populations were used to identify SNP markers closely linked to the SrCad locus. Of 220 SNP markers evaluated, 27 were found to be located within a 2 cM region surrounding SrCad. The diagnostic potential of these SNPs was evaluated in a diverse set of 50 wheat lines that were primarily of Canadian origin with known presence or absence of SrCad. Three SNP markers tightly linked proximally to SrCad and one SNP that co-segregated with SrCad were completely predictive of the presence or absence of SrCad. These markers also differentiated SrCad from Sr42 and SrTmp which are also located in the same region of chromosome arm 6DS. These markers should be useful in marker-assisted breeding to develop new wheat varieties containing SrCad-based resistance to Ug99 stem rust.

A saturated SNP linkage map for the orange wheat blossom midge resistance gene Sm1.

KEY MESSAGE: SNP markers were developed for the OWBM resistance gene Sm1 that will be useful for MAS. The wheat Sm1 region is collinear with an inverted syntenic interval in B. distachyon. Orange wheat blossom midge (OWBM, Sitodiplosis mosellana Géhin) is an important insect pest of wheat (Triticum aestivum) in many growing regions. Sm1 is the only described OWBM resistance gene and is the foundation of managing OWBM through host genetics. Sm1 was previously mapped to wheat chromosome arm 2BS relative to simple sequence repeat (SSR) markers and the dominant, sequence characterized amplified region (SCAR) marker WM1. The objectives of this research were to saturate the Sm1 region with markers, develop improved markers for marker-assisted selection (MAS), and examine the synteny between wheat, Brachypodium distachyon, and rice (Oryza sativa) in the Sm1 region. The present study mapped Sm1 in four populations relative to single nucleotide polymorphisms (SNPs), SSRs, Diversity Array Technology (DArT) markers, single strand conformation polymorphisms (SSCPs), and the SCAR WM1. Numerous high quality SNP assays were designed that mapped near Sm1. BLAST delineated the syntenic intervals in B. distachyon and rice using gene-based SNPs as query sequences. The Sm1 region in wheat was inverted relative to B. distachyon and rice, which suggests a chromosomal rearrangement within the Triticeae lineage. Seven SNPs were tested on a collection of wheat lines known to carry Sm1 and not to carry Sm1. Sm1-flanking SNPs were identified that were useful for predicting the presence or absence of Sm1 based upon haplotype. These SNPs will be a major improvement for MAS of Sm1 in wheat breeding programs.

Synchrotron based phase contrast X-ray imaging combined with FTIR spectroscopy reveals structural and biomolecular differences in spikelets play a significant role in resistance to Fusarium in wheat.

BACKGROUND: Fusarium head blight (FHB), a scab principally caused by Fusarium graminearum Schw., is a serious disease of wheat. The purpose of this study is to evaluate the potential of combining synchrotron based phase contrast X-ray imaging (PCI) with Fourier Transform mid infrared (FTIR) spectroscopy to understand the mechanisms of resistance to FHB by resistant wheat cultivars. Our hypothesis is that structural and biochemical differences between resistant and susceptible cultivars play a significant role in developing resistance to FHB. RESULTS: Synchrotron based PCI images and FTIR absorption spectra (4000-800 cm(-1)) of the floret and rachis from Fusarium-damaged and undamaged spikes of the resistant cultivar 'Sumai3', tolerant cultivar 'FL62R1', and susceptible cultivar 'Muchmore' were collected and analyzed. The PCI images show significant differences between infected and non-infected florets and rachises of different wheat cultivars. However, no pronounced difference between non-inoculated resistant and susceptible cultivar in terms of floret structures could be determined due to the complexity of the internal structures. The FTIR spectra showed significant variability between infected and non-infected floret and rachis of the wheat cultivars. The changes in absorption wavenumbers following pathogenic infection were mostly in the spectral range from 1800-800 cm(-1). The Principal Component Analysis (PCA) was also used to determine the significant chemical changes inside floret and rachis when exposed to the FHB disease stress to understand the plant response mechanism. In the floret and rachis samples, PCA of FTIR spectra revealed differences in cell wall related polysaccharides. In the florets, absorption peaks for Amide I, cellulose, hemicellulose and pectin were affected by the pathogenic fungus. In the rachis of the wheat cultivars, PCA underlines significant changes in pectin, cellulose, and hemicellulose characteristic absorption spectra. Amide II and lignin absorption peaks, persistent in the rachis of Sumai3, together with increased peak shift at 1245 cm(-1) after infection with FHB may be a marker for stress response in which the cell wall compounds related to pathways for lignification are increased. CONCLUSIONS: Synchrotron based PCI combined with FTIR spectroscopy show promising results related to FHB in wheat. The combined technique is a powerful new tool for internal visualisation and biomolecular monitoring before and during plant-microbe interactions to understand both the differences between cultivars and their different responses to disease stress.

A systems genomics and genetics approach to identify the genetic regulatory network for lignin content in Brassica napus seeds.

Seed quality traits of oilseed rape, Brassica napus (B. napus), exhibit quantitative inheritance determined by its genetic makeup and the environment via the mediation of a complex genetic architecture of hundreds to thousands of genes. Thus, instead of single gene analysis, network-based systems genomics and genetics approaches that combine genotype, phenotype, and molecular phenotypes offer a promising alternative to uncover this complex genetic architecture. In the current study, systems genetics approaches were used to explore the genetic regulation of lignin traits in B. napus seeds. Four QTL (qLignin_A09_1, qLignin_A09_2, qLignin_A09_3, and qLignin_C08) distributed on two chromosomes were identified for lignin content. The qLignin_A09_2 and qLignin_C08 loci were homologous QTL from the A and C subgenomes, respectively. Genome-wide gene regulatory network analysis identified eighty-three subnetworks (or modules); and three modules with 910 genes in total, were associated with lignin content, which was confirmed by network QTL analysis. eQTL (expression quantitative trait loci) analysis revealed four cis-eQTL genes including lignin and flavonoid pathway genes, cinnamoyl-CoA-reductase (CCR1), and TRANSPARENT TESTA genes TT4, TT6, TT8, as causal genes. The findings validated the power of systems genetics to identify causal regulatory networks and genes underlying complex traits. Moreover, this information may enable the research community to explore new breeding strategies, such as network selection or gene engineering, to rewire networks to develop climate resilience crops with better seed quality.

High-level expression of sugar inducible gene2 (HSI2) is a negative regulator of drought stress tolerance in Arabidopsis.

BACKGROUND: HIGH-LEVEL EXPRESSION OF SUGAR INDUCIBLE GENE2 (HSI2), also known as VAL1, is a B3 domain transcriptional repressor that acts redundantly with its closest relative, HSI2-LIKE1 (HSL1), to suppress the seed maturation program following germination. Mutant hsi2 hsl1 seedlings are arrested early in development and differentially express a number of abiotic stress-related genes. To test the potential requirement for HSI2 during abiotic stress, hsi2 single mutants and plants overexpressing HSI2 were subjected to simulated drought stress by withholding watering, and characterized through physiological, metabolic and gene expression studies. RESULTS: The hsi2 mutants demonstrated reduced wilting and maintained higher relative water content than wild-type after withholding watering, while the overexpressing lines displayed the opposite phenotype. The hsi2 mutant displayed lower constitutive and ABA-induced stomatal conductance than wild-type and accumulated lower levels of ABA metabolites and several osmolytes and osmoprotectants following water withdrawal. Microarray comparisons between wild-type and the hsi2 mutant revealed that steady-state levels of numerous stress-induced genes were up-regulated in the mutant in the absence of stress but down-regulated at visible wilting. Plants with altered levels of HSI2 responded to exogenous application of ABA and a long-lived ABA analog, but the hsi2 mutant did not show altered expression of several ABA-responsive or ABA signalling genes 4 hr after application. CONCLUSIONS: These results implicate HSI2 as a negative regulator of drought stress response in Arabidopsis, acting, at least in part, by regulating transpirational water loss. Metabolic and global transcript profiling comparisons of the hsi2 mutant and wild-type plants do not support a model whereby the greater drought tolerance observed in the hsi2 mutant is conferred by the accumulation of known osmolytes and osmoprotectants. Instead, data are consistent with mutants experiencing a relatively milder dehydration stress following water withdrawal.

Multi-locus genome-wide association study of fusarium head blight in relation to days to anthesis and plant height in a spring wheat association panel.

Fusarium head blight (FHB) is a highly destructive fungal disease of wheat to which host resistance is quantitatively inherited and largely influenced by the environment. Resistance to FHB has been associated with taller height and later maturity; however, a further understanding of these relationships is needed. An association mapping panel (AMP) of 192 predominantly Canadian spring wheat was genotyped with the wheat 90K single-nucleotide polymorphism (SNP) array. The AMP was assessed for FHB incidence (INC), severity (SEV) and index (IND), days to anthesis (DTA), and plant height (PLHT) between 2015 and 2017 at three Canadian FHB-inoculated nurseries. Seven multi-environment trial (MET) datasets were deployed in a genome-wide association study (GWAS) using a single-locus mixed linear model (MLM) and a multi-locus random SNP-effect mixed linear model (mrMLM). MLM detected four quantitative trait nucleotides (QTNs) for INC on chromosomes 2D and 3D and for SEV and IND on chromosome 3B. Further, mrMLM identified 291 QTNs: 50 (INC), 72 (SEV), 90 (IND), 41 (DTA), and 38 (PLHT). At two or more environments, 17 QTNs for FHB, DTA, and PLHT were detected. Of these 17, 12 QTNs were pleiotropic for FHB traits, DTA, and PLHT on chromosomes 1A, 1D, 2D, 3B, 5A, 6B, 7A, and 7B; two QTNs for DTA were detected on chromosomes 1B and 7A; and three PLHT QTNs were located on chromosomes 4B and 6B. The 1B DTA QTN and the three pleiotropic QTNs on chromosomes 1A, 3B, and 6B are potentially identical to corresponding quantitative trait loci (QTLs) in durum wheat. Further, the 3B pleiotropic QTN for FHB INC, SEV, and IND co-locates with TraesCS3B02G024900 within the Fhb1 region on chromosome 3B and is ~3 Mb from a cloned Fhb1 candidate gene TaHRC. While the PLHT QTN on chromosome 6B is putatively novel, the 1B DTA QTN co-locates with a disease resistance protein located ~10 Mb from a Flowering Locus T1-like gene TaFT3-B1, and the 7A DTA QTN is ~5 Mb away from a maturity QTL QMat.dms-7A.3 of another study. GWAS and QTN candidate genes enabled the characterization of FHB resistance in relation to DTA and PLHT. This approach should eventually generate additional and reliable trait-specific markers for breeding selection, in addition to providing useful information for FHB trait discovery.

Mining biological information from 3D short time-series gene expression data: the OPTricluster algorithm.

BACKGROUND: Nowadays, it is possible to collect expression levels of a set of genes from a set of biological samples during a series of time points. Such data have three dimensions: gene-sample-time (GST). Thus they are called 3D microarray gene expression data. To take advantage of the 3D data collected, and to fully understand the biological knowledge hidden in the GST data, novel subspace clustering algorithms have to be developed to effectively address the biological problem in the corresponding space. RESULTS: We developed a subspace clustering algorithm called Order Preserving Triclustering (OPTricluster), for 3D short time-series data mining. OPTricluster is able to identify 3D clusters with coherent evolution from a given 3D dataset using a combinatorial approach on the sample dimension, and the order preserving (OP) concept on the time dimension. The fusion of the two methodologies allows one to study similarities and differences between samples in terms of their temporal expression profile. OPTricluster has been successfully applied to four case studies: immune response in mice infected by malaria (Plasmodium chabaudi), systemic acquired resistance in Arabidopsis thaliana, similarities and differences between inner and outer cotyledon in Brassica napus during seed development, and to Brassica napus whole seed development. These studies showed that OPTricluster is robust to noise and is able to detect the similarities and differences between biological samples. CONCLUSIONS: Our analysis showed that OPTricluster generally outperforms other well known clustering algorithms such as the TRICLUSTER, gTRICLUSTER and K-means; it is robust to noise and can effectively mine the biological knowledge hidden in the 3D short time-series gene expression data.

Arabidopsis clade I TGA transcription factors regulate plant defenses in an NPR1-independent fashion.

Transcriptional reprogramming during induction of salicylic acid (SA)-mediated defenses is regulated primarily by NPR1 (NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1), likely through interactions with TGA bZIP transcription factors. To ascertain the contributions of clade I TGA factors (TGA1 and TGA4) to defense responses, a tga1-1 tga4-1 double mutant was constructed and challenged with Pseudomonas syringae and Hyaloperonospora arabidopsidis. Although the mutant displayed enhanced susceptibility to virulent P. syringae, it was not compromised in systemic acquired resistance against this pathogen or resistance against avirulent H. arabidopsidis. Microarray analysis of nonelicited and SA-treated plants indicated that clade I TGA factors regulate fewer genes than NPR1. Approximately half of TGA-dependent genes were regulated by NPR1 but, in all cases, the direction of change was opposite in the two mutants. In support of the microarray data, the NPR1-independent disease resistance observed in the autoimmune resistance (R) gene mutant snc1 is partly compromised by tga1-1 tga4-1 mutations, and a triple mutant of clade I TGA factors with npr1-1 is more susceptible than either parent. These results suggest that clade I TGA factors are required for resistance against virulent pathogens and avirulent pathogens mediated by at least some R gene specificities, acting substantially through NPR1-independent pathways.

The BTB/POZ domain of the Arabidopsis disease resistance protein NPR1 interacts with the repression domain of TGA2 to negate its function.

TGA2 and NONEXPRESSER OF PR GENES1 (NPR1) are activators of systemic acquired resistance (SAR) and of the SAR marker gene pathogenesis-related-1 (PR-1) in Arabidopsis thaliana. TGA2 is a transcriptional repressor required for basal repression of PR-1, but during SAR, TGA2 recruits NPR1 as part of an enhanceosome. Transactivation by the enhanceosome requires the NPR1 BTB/POZ domain. However, the NPR1 BTB/POZ domain does not contain an autonomous transactivation domain; thus, its molecular role within the enhanceosome remains elusive. We now show by gel filtration analyses that TGA2 binds DNA as a dimer, tetramer, or oligomer. Using in vivo plant transcription assays, we localize the repression domain of TGA2 to the N terminus and demonstrate that this domain is responsible for modulating the DNA binding activity of the oligomer both in vitro and in vivo. We confirm that the NPR1 BTB/POZ domain interacts with and negates the molecular function of the TGA2 repression domain by excluding TGA2 oligomers from cognate DNA. These data distinguish the NPR1 BTB/POZ domain from other known BTB/POZ domains and establish its molecular role in the context of the Arabidopsis PR-1 gene enhanceosome.

The Seed Coat's Impact on Crop Performance in Pea (Pisum sativum L.).

Seed development in angiosperms produces three genetically and developmentally distinct sub-compartments: the embryo, endosperm, and seed coat. The maternally derived seed coat protects the embryo and interacts closely with the external environment especially during germination and seedling establishment. Seed coat is a key contributor to seed composition and an important determinant of nutritional value for humans and livestock. In this review, we examined pea crop productivity through the lens of the seed coat, its contribution to several valued nutritional traits of the pea crop, and its potential as a breeding target. Key discoveries made in advancing the knowledge base for sensing and transmission of external signals, the architecture and chemistry of the pea seed coat, and relevant insights from other important legumes were discussed. Furthermore, for selected seed coat traits, known mechanisms of genetic regulation and efforts to modulate these mechanisms to facilitate composition and productivity improvements in pea were discussed, alongside opportunities to support the continued development and improvement of this underutilized crop. This review describes the most important features of seed coat development in legumes and highlights the key roles played by the seed coat in pea seed development, with a focus on advances made in the genetic and molecular characterization of pea and other legumes and the potential of this key seed tissue for targeted improvement and crop optimization.

Localization of DIR1 at the tissue, cellular and subcellular levels during Systemic Acquired Resistance in Arabidopsis using DIR1:GUS and DIR1:EGFP reporters.

BACKGROUND: Systemic Acquired Resistance (SAR) is an induced resistance response to pathogens, characterized by the translocation of a long-distance signal from induced leaves to distant tissues to prime them for increased resistance to future infection. DEFECTIVE in INDUCED RESISTANCE 1 (DIR1) has been hypothesized to chaperone a small signaling molecule to distant tissues during SAR in Arabidopsis. RESULTS: DIR1 promoter:DIR1-GUS/dir1-1 lines were constructed to examine DIR1 expression. DIR1 is expressed in seedlings, flowers and ubiquitously in untreated or mock-inoculated mature leaf cells, including phloem sieve elements and companion cells. Inoculation of leaves with SAR-inducing avirulent or virulent Pseudomonas syringae pv tomato (Pst) resulted in Type III Secretion System-dependent suppression of DIR1 expression in leaf cells. Transient expression of fluorescent fusion proteins in tobacco and intercellular washing fluid experiments indicated that DIR1's ER signal sequence targets it for secretion to the cell wall. However, DIR1 expressed without a signal sequence rescued the dir1-1 SAR defect, suggesting that a cytosolic pool of DIR1 is important for the SAR response. CONCLUSIONS: Although expression of DIR1 decreases during SAR induction, the protein localizes to all living cell types of the vasculature, including companion cells and sieve elements, and therefore DIR1 is well situated to participate in long-distance signaling during SAR.

Arabidopsis basic leucine-zipper transcription factors TGA9 and TGA10 interact with floral glutaredoxins ROXY1 and ROXY2 and are redundantly required for anther development.

ROXY1 and ROXY2 are CC-type floral glutaredoxins with redundant functions in Arabidopsis (Arabidopsis thaliana) anther development. We show here that plants lacking the basic leucine-zipper transcription factors TGA9 and TGA10 have defects in male gametogenesis that are strikingly similar to those in roxy1 roxy2 mutants. In tga9 tga10 mutants, adaxial and abaxial anther lobe development is differentially affected, with early steps in anther development blocked in adaxial lobes and later steps affected in abaxial lobes. Distinct from roxy1 roxy2, microspore development in abaxial anther lobes proceeds to a later stage with the production of inviable pollen grains contained within nondehiscent anthers. Histological analysis shows multiple defects in the anther dehiscence program, including abnormal stability and lignification of the middle layer and defects in septum and stomium function. Compatible with these defects, TGA9 and TGA10 are expressed throughout early anther primordia but resolve to the middle and tapetum layers during meiosis of pollen mother cells. Several lines of evidence suggest that ROXY promotion of anther development is mediated in part by TGA9 and TGA10. First, TGA9 and TGA10 expression overlaps with ROXY1/2 during anther development. Second, TGA9/10 and ROXY1/2 operate downstream of SPOROCYTELESS/NOZZLE, where they positively regulate a common set of genes that contribute to tapetal development. Third, TGA9 and TGA10 directly interact with ROXY proteins in yeast and in plant cell nuclei. These findings suggest that activation of TGA9/10 transcription factors by ROXY-mediated modification of cysteine residues promotes anther development, thus broadening our understanding of how redox-regulated TGA factors function in plants.