Evolution of the Grain Dispersal System in Barley.

About 12,000 years ago in the Near East, humans began the transition from hunter-gathering to agriculture-based societies. Barley was a founder crop in this process, and the most important steps in its domestication were mutations in two adjacent, dominant, and complementary genes, through which grains were retained on the inflorescence at maturity, enabling effective harvesting. Independent recessive mutations in each of these genes caused cell wall thickening in a highly specific grain "disarticulation zone," converting the brittle floral axis (the rachis) of the wild-type into a tough, non-brittle form that promoted grain retention. By tracing the evolutionary history of allelic variation in both genes, we conclude that spatially and temporally independent selections of germplasm with a non-brittle rachis were made during the domestication of barley by farmers in the southern and northern regions of the Levant, actions that made a major contribution to the emergence of early agrarian societies.

Wheat Resistances to Fusarium Root Rot and Head Blight Are Both Associated with Deoxynivalenol- and Jasmonate-Related Gene Expression.

Fusarium graminearum is a major pathogen of wheat causing Fusarium head blight (FHB). Its ability to colonize wheat via seedling root infection has been reported recently. Our previous study on Fusarium root rot (FRR) has disclosed histological characteristics of pathogenesis and pathogen defense that mirror processes of spike infection. Therefore, it would be interesting to understand whether genes relevant for FHB resistance are induced in roots. The concept of similar-acting defense mechanisms provides a basis for research at broad Fusarium resistance in crop plants. However, molecular defense responses involved in FRR as well as their relation to spike resistance are unknown. To test the hypothesis of a conserved defense response, a candidate gene expression study was conducted to test the activity of selected prominent FHB defense-related genes in seedling roots, adult plant roots, spikes, and shoots. FRR was examined at seedling and adult plant stages to assess age-related pattern of disease and pathogen resistance. This study offers first evidence for a significant genetic overlap in root and spike defense responses, both in local and distant tissues. The results point to plant development-specific rather than organ-specific determinants of resistance, and suggest roots as an interesting model for studies on wheat-Fusarium interactions.

Insights Into Triticum aestivum Seedling Root Rot Caused by Fusarium graminearum.

Fusarium graminearum is one of the most common and potent fungal pathogens of wheat (Triticum aestivum), known for causing devastating spike infections and grain yield damage. F. graminearum is a typical soil-borne pathogen that builds up during consecutive cereal cropping. Speculation on systemic colonization of cereals by F. graminearum root infection have long existed but have not been proven. We have assessed the Fusarium root rot disease macroscopically in a diverse set of 12 wheat genotypes and microscopically in a comparative study of two genotypes with diverging responses. Here, we show a 'new' aspect of the F. graminearum life cycle, i.e., the head blight fungus uses a unique root-infection strategy with an initial stage typical for root pathogens and a later stage typical for spike infection. Root colonization negatively affects seedling development and leads to systemic plant invasion by tissue-adapted fungal strategies. Another major outcome is the identification of partial resistance to root rot. Disease severity assessments and histological examinations both demonstrated three distinct disease phases that, however, proceeded differently in resistant and susceptible genotypes. Soil-borne inoculum and root infection are considered significant components of the F. graminearum life cycle with important implications for the development of new strategies of resistance breeding and disease control.

High resolution mass spectrometry imaging of plant tissues: towards a plant metabolite atlas.

Mass spectrometry (MS) imaging provides spatial and molecular information for a wide range of compounds. This tool can be used to investigate metabolic changes in plant physiology and environmental interactions. A major challenge in our study was to prepare tissue sections that were compatible with high spatial resolution analysis and therefore dedicated sample preparation protocols were established and optimized for the physicochemical properties of all major plant organs. We combined high spatial resolution (5 µm), in order to detect cellular features, and high mass accuracy (<2 ppm root mean square error), for molecular specificity. Mass spectrometry imaging experiments were performed in positive and negative ion mode. Changes in metabolite patterns during plant development were investigated for germination of oilseed rape. The detailed localization of more than 90 compounds allowed assignment to metabolic processes and indicated possible functions in plant tissues. The 'untargeted' nature of MS imaging allows the detection of marker compounds for the physiological status, as demonstrated for plant-pathogen interactions. Our images show excellent correlation with optical/histological examination. In contrast to previous MS imaging studies of plants, we present a complete workflow that covers multiple species, such as oilseed rape, wheat seed and rice. In addition, different major plant organs and a wide variety of compound classes were analyzed. Thus, our method could be used to develop a plant metabolite atlas as a reference to investigate systemic and local effects of pathogen infection or environmental stress.

Jasmonate and ethylene dependent defence gene expression and suppression of fungal virulence factors: two essential mechanisms of Fusarium head blight resistance in wheat?

BACKGROUND: Fusarium head blight (FHB) caused by Fusarium species like F. graminearum is a devastating disease of wheat (Triticum aestivum) worldwide. Mycotoxins such as deoxynivalenol produced by the fungus affect plant and animal health, and cause significant reductions of grain yield and quality. Resistant varieties are the only effective way to control this disease, but the molecular events leading to FHB resistance are still poorly understood. Transcriptional profiling was conducted for the winter wheat cultivars Dream (moderately resistant) and Lynx (susceptible). The gene expressions at 32 and 72 h after inoculation with Fusarium were used to trace possible defence mechanisms and associated genes. A comparative qPCR was carried out for selected genes to analyse the respective expression patterns in the resistant cultivars Dream and Sumai 3 (Chinese spring wheat). RESULTS: Among 2,169 differentially expressed genes, two putative main defence mechanisms were found in the FHB-resistant Dream cultivar. Both are defined base on their specific mode of resistance. A non-specific mechanism was based on several defence genes probably induced by jasmonate and ethylene signalling, including lipid-transfer protein, thionin, defensin and GDSL-like lipase genes. Additionally, defence-related genes encoding jasmonate-regulated proteins were up-regulated in response to FHB. Another mechanism based on the targeted suppression of essential Fusarium virulence factors comprising proteases and mycotoxins was found to be an essential, induced defence of general relevance in wheat. Moreover, similar inductions upon fungal infection were frequently observed among FHB-responsive genes of both mechanisms in the cultivars Dream and Sumai 3. CONCLUSIONS: Especially ABC transporter, UDP-glucosyltransferase, protease and protease inhibitor genes associated with the defence mechanism against fungal virulence factors are apparently active in different resistant genetic backgrounds, according to reports on other wheat cultivars and barley. This was further supported in our qPCR experiments on seven genes originating from this mechanism which revealed similar activities in the resistant cultivars Dream and Sumai 3. Finally, the combination of early-stage and steady-state induction was associated with resistance, while transcript induction generally occurred later and temporarily in the susceptible cultivars. The respective mechanisms are attractive for advanced studies aiming at new resistance and toxin management strategies.

Mass Spectrometry Imaging Disclosed Spatial Distribution of Defense-Related Metabolites in Triticum spp.

Fusarium Head Blight is the most common fungal disease that strongly affects Triticum spp., reducing crop yield and leading to the accumulation of toxic metabolites. Several studies have investigated the plant metabolic response to counteract mycotoxins accumulation. However, information on the precise location where the defense mechanism is taking place is scarce. Therefore, this study aimed to investigate the specific tissue distribution of defense metabolites in two Triticum species and use this information to postulate on the metabolites' functional role, unlocking the "location-to-function" paradigm. To address this challenge, transversal cross-sections were obtained from the middle of the grains. They were analyzed using an atmospheric-pressure (AP) SMALDI MSI source (AP-SMALDI5 AF, TransMIT GmbH, Giessen, Germany) coupled to a Q Exactive HF (Thermo Fisher Scientific GmbH, Bremen, Germany) orbital trapping mass spectrometer. Our result revealed the capability of (AP)-SMALDI MSI instrumentation to finely investigate the spatial distribution of wheat defense metabolites, such as hydroxycinnamic acid amides, oxylipins, linoleic and α-linoleic acids, galactolipids, and glycerolipids.

Histology-guided high-resolution AP-SMALDI mass spectrometry imaging of wheat-Fusarium graminearum interaction at the root-shoot junction.

BACKGROUND: Fungal pathogens like Fusarium graminearum can cause severe yield losses and mycotoxin contamination of food and feed worldwide. We recently showed its ability to systemically colonize wheat via root infection. However, the molecular response of wheat to Fusarium root rot (FRR) infection and systemic spread is still unknown. As a molecular camera, mass spectrometry (MS) imaging combines label-free and multiplex metabolite profiling with histopathology. RESULTS: Atmospheric-pressure (AP)-SMALDI-MS imaging was combined with optical microscopy to study wheat-F. graminearum interaction at the root-shoot junction, which is a crucial line of defense against a pathogen that can invade all distal plant parts. To scope the functional, temporal and local aspects of FRR disease spread, metabolic changes were simultaneous visualized in diseased and healthy stem bases of the resistant cultivar Florence-Aurore at 10, 14 and 21 days after root inoculation. Histological information was used to identify disease relevant tissues and to assist the interpretation of molecular images. Detected mycotoxin compounds secreted by F. graminearum showed a route of stem infection that was consistent with observations made by microscopy. The outer epidermis and vasculature of leaf sheath were, at different disease stages, identified as prominent sites of pathogen migration and wheat protection. Wheat metabolites mapped to these relatively small tissues indicated cell wall strengthening and antifungal activity as direct defenses as well as conservation in the wheat reactions to F. graminearum diseases that affect different plant organs. CONCLUSIONS: AP-SMALDI-MS imaging at high spatial resolution is a versatile technique that can be applied to basic and applied aspects of agricultural research. Combining the technology with optical microscopy was found to be a powerful tool to gain in-depth information on almost unknown crop disease. Moreover, the approach allowed studying metabolism at the host-pathogen interface. The results provide important hints to an understanding of the complex spatio-temporal organization of plant resistance. Defense-on-demand responses to pathogen ingress were found, which provide opportunities for future research towards an improved resistance that does not negatively impact yield development in the field by saving plant resources and, moreover, may control different Fusarium diseases.

Genetic insights into underground responses to Fusarium graminearum infection in wheat.

The ongoing global intensification of wheat production will likely be accompanied by a rising pressure of Fusarium diseases. While utmost attention was given to Fusarium head blight (FHB) belowground plant infections of the pathogen have largely been ignored. The current knowledge about the impact of soil borne Fusarium infection on plant performance and the underlying genetic mechanisms for resistance remain very limited. Here, we present the first large-scale investigation of Fusarium root rot (FRR) resistance using a diverse panel of 215 international wheat lines. We obtained data for a total of 21 resistance-related traits, including large-scale Real-time PCR experiments to quantify fungal spread. Association mapping and subsequent haplotype analyses discovered a number of highly conserved genomic regions associated with resistance, and revealed a significant effect of allele stacking on the stembase discoloration. Resistance alleles were accumulated in European winter wheat germplasm, implying indirect prior selection for improved FRR resistance in elite breeding programs. Our results give first insights into the genetic basis of FRR resistance in wheat and demonstrate how molecular parameters can successfully be explored in genomic prediction. Ongoing work will help to further improve our understanding of the complex interactions of genetic factors influencing FRR resistance.

Wheat Root-dip Inoculation with Fusarium graminearum and Assessment of Root Rot Disease Severity.

Fusarium graminearum is one of the most common and potent fungal pathogens of wheat (Triticum aestivum) and other cereals, known for causing devastating yield losses and mycotoxin contaminations of food and feed. The pathogen is mainly considered as a paradigm for the floral disease Fusarium head blight, while its ability to colonize wheat plants via root infection has been examined recently. F. graminearum has a unique infection strategy which comprises complex, specialized structures and processes. Root colonisation negatively affects plant development and leads to systemic plant invasion by tissue-adapted fungal strategies. The pathosystem wheat root - F. graminearum makes available an array of research areas, such as (i) the relatively unknown root interactions with a necrotrophic pathogen; (ii) genes and pathways contributing to (overall) Fusarium resistance; (iii) induced systemic (whole-plant) resistance; (iv) pathogenic strategies in a variety of host tissues; and (v) age-related changes in the single-genotype responses to seedling and adult plant (root/spike) infection. The presented Fusarium root rot bioassay allows for efficient infection of wheat roots, evaluation of disease severity and progress as well as statistical analysis of disease dynamics.

Subgenomic Diversity Patterns Caused by Directional Selection in Bread Wheat Gene Pools.

Genetic diversity represents the fundamental key to breeding success, providing the basis for breeders to select varieties with constantly improving yield performance. On the other hand, strong selection during domestication and breeding have eliminated considerable genetic diversity in the breeding pools of major crops, causing erosion of genetic potential for adaptation to emerging challenges like climate change. High-throughput genomic technologies can address this dilemma by providing detailed knowledge to characterize and replenish genetic diversity in breeding programs. In hexaploid bread wheat (Triticum aestivum L.), the staple food for 35% of the world's population, bottlenecks during allopolyploidisation followed by strong artificial selection have considerably narrowed diversity to the extent that yields in many regions appear to be unexpectedly stagnating. In this study, we used a 90,000 single nucleotide polymorphism (SNP) wheat genotyping array to assay high-frequency, polymorphic SNP markers in 460 accessions representing different phenological diversity groups from Asian, Australian, European, and North American bread wheat breeding materials. Detailed analysis of subgroup diversity at the chromosome and subgenome scale revealed highly distinct patterns of conserved linkage disequilibrium between different gene pools. The data enable identification of genome regions in most need of rejuvenation with novel diversity and provide a high-resolution molecular basis for genomic-assisted introgression of new variation into chromosome segments surrounding directionally selected metaloci conferring important adaptation and quality traits.

TILLING in the two-rowed barley cultivar 'Barke' reveals preferred sites of functional diversity in the gene HvHox1.

BACKGROUND: The economic importance of cereals such as barley, and the demand for improved yield and quality require a better understanding of the genetic components that modulate biologically and commercially relevant traits. While Arabidopsis thaliana is the premiere model plant system, the spectrum of its traits cannot address all of the fundamental questions of crop plant development. Unlike Arabidopsis, barley is both a crop and a model system for scientific research, and it is increasingly being used for genetic and molecular investigations into the conserved biological processes of cereals. A common challenge in genetic studies in plants with large genomes arises from the very time-consuming work of associating mutant phenotypes with gene sequence information, especially if insertion mutagenesis is not routine, as in barley. Reverse genetics based on chemical mutagenesis represents the best solution to this obstacle. FINDINGS: In barley, we generated a new TILLING (Targeting Local Lesions IN Genomes) resource comprising 10,279 M(2 )mutants in the two-rowed malting cultivar 'Barke,' which has been used in the generation of other genomic resources in barley (~150,000 ESTs, DH mapping population). The value of this new resource was tested using selected candidate genes. An average frequency of approximately one mutation per 0.5 Mb was determined by screening ten fragments of six different genes. The ethyl methanesulphonate (EMS)mutagenesis efficiency was studied by recording and relating the mutagenesis-dependent effects found in the three mutant generations (M(1)-M(3)). A detailed analysis was performed for the homeodomain-leucine-zipper (HD-ZIP) gene HvHox1. Thirty-one mutations were identified by screening a 1,270-bp fragment in 7,348 M(2 )lines. Three of the newly identified mutants exhibited either a six-rowed or an intermedium-spike phenotype, and one mutant displayed a significantly altered spikelet morphology compared to that of the 'Barke' wild type. Our results indicate a bias in the frequency of independent functional mutations at specific base pair (bp) positions within the gene HvHox1. CONCLUSIONS: A new TILLING population was developed as a resource for high-throughput gene discovery in an alternative barley germplasm. Pilot screening demonstrated a similar or even slightly higher mutation frequency when compared to previously published barley TILLING populations that should allow for the identification of diverse allelic variation. Partial phenotypic evaluation of the M(2 )and M(3 )generations has revealed the presence of a wide spectrum of morphological diversity that highlights the great potential of this resource for use in forward genetic screens. Altogether, our study shows the efficiency of screening and the applicability of the new TILLING population for genetic studies in the barley crop model system.