Gene expression evolution in pattern-triggered immunity within Arabidopsis thaliana and across Brassicaceae species.

Plants recognize surrounding microbes by sensing microbe-associated molecular patterns (MAMPs) to activate pattern-triggered immunity (PTI). Despite their significance for microbial control, the evolution of PTI responses remains largely uncharacterized. Here, by employing comparative transcriptomics of six Arabidopsis thaliana accessions and three additional Brassicaceae species to investigate PTI responses, we identified a set of genes that commonly respond to the MAMP flg22 and genes that exhibit species-specific expression signatures. Variation in flg22-triggered transcriptome responses across Brassicaceae species was incongruent with their phylogeny, while expression changes were strongly conserved within A. thaliana. We found the enrichment of WRKY transcription factor binding sites in the 5'-regulatory regions of conserved and species-specific responsive genes, linking the emergence of WRKY-binding sites with the evolution of gene expression patterns during PTI. Our findings advance our understanding of the evolution of the transcriptome during biotic stress.

Phloem Companion Cell-Specific Transcriptomic and Epigenomic Analyses Identify MRF1, a Regulator of Flowering.

The phloem plays essential roles in the source-to-sink relationship and in long-distance communication, and thereby coordinates growth and development throughout the plant. Here we employed isolation of nuclei tagged in specific cell types coupled with low-input, high-throughput sequencing approaches to analyze the changes of the chromatin modifications H3K4me3 and H3K27me3 and their correlation with gene expression in the phloem companion cells (PCCs) of Arabidopsis(Arabidopsis thaliana) shoots in response to changes in photoperiod. We observed a positive correlation between changes in expression and H3K4me3 levels of genes that are involved in essential PCC functions, including regulation of metabolism, circadian rhythm, development, and epigenetic modifications. By contrast, changes in H3K27me3 signal appeared to contribute little to gene expression changes. These genomic data illustrate the complex gene-regulatory networks that integrate plant developmental and physiological processes in the PCCs. Emphasizing the importance of cell-specific analyses, we identified a previously uncharacterized MORN-motif repeat protein, MORN-MOTIF REPEAT PROTEIN REGULATING FLOWERING1 (MRF1), that was strongly up-regulated in the PCCs in response to inductive photoperiod. The mrf1 mutation delayed flowering, whereas MRF1 overexpression had the opposite effect, indicating that MRF1 acts as a floral promoter.

Targeted and Untargeted Metabolomic Analyses Reveal Organ Specificity of Specialized Metabolites in the Model Grass Brachypodium distachyon.

Brachypodium distachyon, because of its fully sequenced genome, is frequently used as a model grass species. However, its metabolome, which constitutes an indispensable element of complex biological systems, remains poorly characterized. In this study, we conducted comprehensive, liquid chromatography-mass spectrometry (LC-MS)-based metabolomic examination of roots, leaves and spikes of Brachypodium Bd21 and Bd3-1 lines. Our pathway enrichment analysis emphasised the accumulation of specialized metabolites representing the flavonoid biosynthetic pathway in parallel with processes related to nucleotide, sugar and amino acid metabolism. Similarities in metabolite profiles between both lines were relatively high in roots and leaves while spikes showed higher metabolic variance within both accessions. In roots, differences between Bd21 and Bd3-1 lines were manifested primarily in diterpenoid metabolism, while differences within spikes and leaves concerned nucleotide metabolism and nitrogen management. Additionally, sulphate-containing metabolites differentiated Bd21 and Bd3-1 lines in spikes. Structural analysis based on MS fragmentation spectra enabled identification of 93 specialized metabolites. Among them phenylpropanoids and flavonoids derivatives were mainly determined. As compared with closely related barley and wheat species, metabolic profile of Brachypodium is characterized with presence of threonate derivatives of hydroxycinnamic acids.

Phenolic Metabolites from Barley in Contribution to Phenome in soil Moisture Deficit.

Eight barley varieties from Europe and Asia were subjected to moisture deficit at various development stages. At the seedling stage and the flag leaf stage combined stress was applied. The experiment was designed for visualization of the correlation between the dynamics of changes in phenolic compound profiles and the external phenome. The most significant increase of compound content in water deficiency was observed for chrysoeriol and apigenin glycoconjugates acylated with methoxylated hydroxycinnamic acids that enhanced the UV-protection effectiveness. Moreover, other good antioxidants such as derivatives of luteolin and hordatines were also induced by moisture deficit. The structural diversity of metabolites of the contents changed in response to water deficiency in barley indicates their multipath activities under stress. Plants exposed to moisture deficit at the seedling stage mobilized twice as many metabolites as plants exposed to this stress at the flag leaf stage. Specific metabolites such as methoxyhydroxycinnamic acids participated in the long-term acclimation. In addition, differences in phenolome mobilization in response to moisture deficit applied at the vegetative and generative phases were correlated with the phenotypical consequences. Observations of plant yield and biomass gave us the possibility to discuss the developmentally related consequences of moisture deficit for plants' fitness.

Identification of drought responsive proteins and related proteomic QTLs in barley.

Drought is a major abiotic stress that negatively influences crop yield. Breeding strategies for improved drought resistance require an improved knowledge of plant drought responses. We therefore applied drought to barley recombinant inbred lines and their parental genotypes shortly before tillering. A large-scale proteomic analysis of leaf and root tissue revealed proteins that respond to drought in a genotype-specific manner. Of these, Rubisco activase in chloroplast, luminal binding protein in endoplasmic reticulum, phosphoglycerate mutase, glutathione S-transferase, heat shock proteins and enzymes involved in phenylpropanoid biosynthesis showed strong genotype×environment interactions. These data were subjected to genetic linkage analysis and the identification of proteomic QTLs that have potential value in marker-assisted breeding programs.

Drought-related secondary metabolites of barley (Hordeum vulgare L.) leaves and their metabolomic quantitative trait loci.

Determining the role of plant secondary metabolites in stress conditions is problematic due to the diversity of their structures and the complexity of their interdependence with different biological pathways. Correlation of metabolomic data with the genetic background provides essential information about the features of metabolites. LC-MS analysis of leaf metabolites from 100 barley recombinant inbred lines (RILs) revealed that 98 traits among 135 detected phenolic and terpenoid compounds significantly changed their level as a result of drought stress. Metabolites with similar patterns of change were grouped in modules, revealing differences among RILs and parental varieties at early and late stages of drought. The most significant changes in stress were observed for ferulic and sinapic acid derivatives as well as acylated glycosides of flavones. The tendency to accumulate methylated compounds was a major phenomenon in this set of samples. In addition, the polyamine derivatives hordatines as well as terpenoid blumenol C derivatives were observed to be drought related. The correlation of drought-related compounds with molecular marker polymorphisms resulted in the definition of metabolomic quantitative trait loci in the genomic regions of single-nucleotide polymorphism 3101-111 and simple sequence repeat Bmag0692 with multiple linkages to metabolites. The associations pointed to genes related to the defence response and response to cold, heat and oxidative stress, but not to genes related to biosynthesis of the compounds. We postulate that the significant metabolites have a role as antioxidants, regulators of gene expression and modulators of protein function in barley during drought.

Simultaneous selection for yield-related traits and susceptibility to Fusarium head blight in spring wheat RIL population.

Fusarium head blight (FHB), caused by the fungal plant pathogen Fusarium, is a fungal disease that occurs in wheat and can cause significant yield and grain quality losses. The present paper examines variation in the resistance of spring wheat lines derived from a cross between Zebra and Saar cultivars. Experiments covering 198 lines and parental cultivars were conducted in three years, in which inoculation with Fusarium culmorum was applied. Resistance levels were estimated by scoring disease symptoms on kernels. In spite of a similar reaction of parents to F. culmorum infection, significant differentiation between lines was found in all the analyzed traits. Seven molecular markers selected as linked to FHB resistance QTLs gave polymorphic products for Zebra and Saar: Xgwm566, Xgwm46, Xgwm389, Xgwm533, Xgwm156, Xwmc238, and Xgwm341. Markers Xgwm389 and Xgwm533 were associated with the rate of Fusarium-damaged kernels (FDK) as well as with kernel weight per spike and thousand kernel weight in control plants. Zebra allele of marker Xwmc238 increased kernel weight per spike and thousand kernel weight both in control and infected plants, whereas Zebra allele of marker Xgwm566 reduced the percentage of FDK and simultaneously reduced the thousand kernel weight in control and infected plants.

Metabolomic Aspects of Conservative and Resistance-Related Elements of Response to Fusarium culmorum in the Grass Family.

BACKGROUND: Fusarium head blight (FHB) is a serious fungal disease affecting crop plants, causing substantial yield reductions and the production of mycotoxins in the infected grains. Achieving progress in the breeding of crops with increased resistance and maintaining a high yield is not possible without a thorough examination of the molecular basis of plant immunity responses. METHODS: LC-MS-based metabolomics approaches powered by three-way ANOVA and the selec-tion of differentially accumulated metabolites (DAMs) were used for studying plant immunity. A correlation network and functional enrichment analysis were conducted on grains of barley and wheat genotypes that were resistant or susceptible to FHB, as well as on the model grass Brachypodium distachyon (Bd), as this is still poorly understood at the metabolomic level. RESULTS: We selected common and genotype-specific DAMs in response to F. culmorum inoculation. The immunological reaction at the metabolomic level was strongly diversified between resistant and susceptible genotypes. DAMs that were common to all tested species from the porphyrin, flavonoid, and phenylpropanoid metabolic pathways were highly correlated, reflecting con-servativeness in the FHB response in the Poaceae family. Resistance-related DAMs belonged to different structural classes, including tryptophan-derived metabolites, pyrimidines, the amino acids proline and serine, as well as phenylpropanoids and flavonoids. The physiological re-sponse to F. culmorum of Bd was close to that of barley and wheat genotypes; however, metabo-lomic changes were strongly diversified. CONCLUSIONS: Combined targeted and untargeted metabolomics provides comprehensive knowledge about significant elements of plant immuni-ty that have the potential to be molecular biomarkers of enhanced resistance to FHB in the grass family. Thorough examination of the Bd metabolome in juxtaposition with diversified geno-types of barley and wheat facilitated its use as a model grass for plant-microbe interaction.

Separation of Chromatographic Co-Eluted Compounds by Clustering and by Functional Data Analysis.

Peak overlapping is a common problem in chromatography, mainly in the case of complex biological mixtures, i.e., metabolites. Due to the existence of the phenomenon of co-elution of different compounds with similar chromatographic properties, peak separation becomes challenging. In this paper, two computational methods of separating peaks, applied, for the first time, to large chromatographic datasets, are described, compared, and experimentally validated. The methods lead from raw observations to data that can form inputs for statistical analysis. First, in both methods, data are normalized by the mass of sample, the baseline is removed, retention time alignment is conducted, and detection of peaks is performed. Then, in the first method, clustering is used to separate overlapping peaks, whereas in the second method, functional principal component analysis (FPCA) is applied for the same purpose. Simulated data and experimental results are used as examples to present both methods and to compare them. Real data were obtained in a study of metabolomic changes in barley (Hordeum vulgare) leaves under drought stress. The results suggest that both methods are suitable for separation of overlapping peaks, but the additional advantage of the FPCA is the possibility to assess the variability of individual compounds present within the same peaks of different chromatograms.

Silver nanoparticles affect phenolic and phytoalexin composition of Arabidopsis thaliana.

Silver nanoparticles are widely used in industry, medicine, biotechnology and agriculture. As a consequence, these nanoparticles are reaching the environment as waste products, which might have a negative impact on the environment, especially on plants. This includes the elicitation of various biochemical processes in plants. In this article, we report on the changes in secondary metabolic profile of Arabidopsis thaliana seedlings subjected to silver nanoparticle treatment in vitro. Briefly, various sizes (10 nm, 40 nm and 100 nm in diameter) and concentrations (0.5-5.0 ppm) of silver nanoparticles were tested. Ultraperformance liquid chromatography coupled with ultraviolet and fluorescence detectors as well as hyphenated to a high-resolution mass spectrometer (UPLC-PDA-FLR, UPLC-HESI-HRMS) and HPLC - ion trap mass spectrometer (HPLC-ESI-MS/MS), were applied to identify and quantify secondary metabolites. To understand whether silver ions could induce changes in the secondary metabolite profile, seedlings treated with silver nitrate in concentrations equivalent to these of nanoparticles were also analysed. The results showed significant differences in the accumulation of phenolic and indole compounds between treatments. Silver nanoparticles and silver ions induced the biosynthesis of camalexin, hydroxycamalexin O-hexoside and hydroxycamalexin malonyl-hexoside. These compounds are important phytoalexins for Brassicaceae family (especially for Camelinae clad) and are also synthetized in response to biotic and abiotic stresses. Statistically significant changes have been also observed for five phenolic compounds and 5'Glucosyl-dihydroneoascorbigen in different treatment conditions.

QTLs for earliness and yield-forming traits in the Lubuski × CamB barley RIL population under various water regimes.

Drought has become more frequent in Central Europe causing large losses in cereal yields, especially of spring crops. The development of new varieties with increased tolerance to drought is a key tool for improvement of agricultural productivity. Material for the study consisted of 100 barley recombinant inbred lines (RILs) (LCam) derived from the cross between Syrian and European parents. The RILs and parental genotypes were examined in greenhouse experiments under well-watered and water-deficit conditions. During vegetation the date of heading, yield and yield-related traits were measured. RIL population was genotyped with microsatellite and single nucleotide polymorphism markers. This population, together with two other populations, was the basis for the consensus map construction, which was used for identification of quantitative trait loci (QTLs) affecting the traits. The studied lines showed a large variability in heading date. It was noted that drought-treatment negatively affected the yield and its components, especially when applied at the flag leaf stage. In total, 60 QTLs were detected on all the barley chromosomes. The largest number of QTLs was found on chromosome 2H. The main QTL associated with heading, located on chromosome 2H (Q.HD.LC-2H), was identified at SNP marker 5880-2547, in the vicinity of Ppd-H1 gene. SNP 5880-2547 was also the closest marker to QTLs associated with plant architecture, spike morphology and grain yield. The present study showed that the earliness allele from the Syrian parent, as introduced into the genome of an European variety could result in an improvement of barley yield performance under drought conditions.

Quantitative trait loci for plant height in Maresi × CamB barley population and their associations with yield-related traits under different water regimes.

High-yielding capacity of the modern barley varieties is mostly dependent on the sources of semi-dwarfness associated with the sdw1/denso locus. The objective of the study was to identify quantitative trait loci (QTLs) associated with the plant height and yield potential of barley recombinant inbred lines (RILs) grown under various soil moisture regimes. The plant material was developed from a hybrid between the Maresi (European cv.) and CamB (Syrian cv.). A total of 103 QTLs affecting analysed traits were detected and 36 of them showed stable effects over environments. In total, ten QTLs were found to be significant only under water shortage conditions. Nine QTLs affecting the length of main stem were detected on 2H-6H chromosomes. In four of the detected QTLs, alleles contributed by Maresi had negative effects on that trait, the most significant being the QLSt-3H.1-1 in the 3H.1 linkage group. The close linkage between QTLs identified around the sdw1/denso locus, with positive alleles contributed by Maresi, indicates that the semi-dwarf cv. Maresi could serve as a donor of favourable traits resulting in grain yield improvement, also under water scarcity. Molecular analyses revealed that the Syrian cv. also contributed alleles which increased the yield potential. Available barley resources of genomic annotations were employed to the biological interpretation of detected QTLs. This approach revealed 26 over-represented Gene Ontology terms. In the projected support intervals of QGWSl-5H.3-2 and QLSt-5H.3 on the chromosome 5H, four genes annotated to 'response to stress' were found. It suggests that these QTL-regions may be involved in a response of plant to a wide range of environmental disturbances.

Temporal dynamics of gene expression and histone marks at the Arabidopsis shoot meristem during flowering.

Plants can produce organs throughout their entire life from pluripotent stem cells located at their growing tip, the shoot apical meristem (SAM). At the time of flowering, the SAM of Arabidopsis thaliana switches fate and starts producing flowers instead of leaves. Correct timing of flowering in part determines reproductive success, and is therefore under environmental and endogenous control. How epigenetic regulation contributes to the floral transition has eluded analysis so far, mostly because of the poor accessibility of the SAM. Here we report the temporal dynamics of the chromatin modifications H3K4me3 and H3K27me3 and their correlation with transcriptional changes at the SAM in response to photoperiod-induced flowering. Emphasizing the importance of tissue-specific epigenomic analyses we detect enrichments of chromatin states in the SAM that were not apparent in whole seedlings. Furthermore, our results suggest that regulation of translation might be involved in adjusting meristem function during the induction of flowering.

Remodeling of Leaf Cellular Glycerolipid Composition under Drought and Re-hydration Conditions in Grasses from the Lolium-Festuca Complex.

Drought tolerant plant genotypes are able to maintain stability and integrity of cellular membranes in unfavorable conditions, and to regenerate damaged membranes after stress cessation. The profiling of cellular glycerolipids during drought stress performed on model species such as Arabidopsis thaliana does not fully cover the picture of lipidome in monocots, including grasses. Herein, two closely related introgression genotypes of Lolium multiflorum (Italian ryegrass) × Festuca arundinacea (tall fescue) were used as a model for other grass species to describe lipid rearrangements during drought and re-hydration. The genotypes differed in their level of photosynthetic capacity during drought, and in their capacity for membrane regeneration after stress cessation. A total of 120 lipids, comprising the classes of monogalactosyldiacyloglycerol, digalactosyldiacyloglycerol, sulfoquinovosyldiacylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, diacylglicerol, and triacylglicerol, were analyzed. The results clearly showed that water deficit had a significant impact on lipid metabolism in studied forage grasses. It was revealed that structural and metabolic lipid species changed their abundance during drought and re-watering periods and some crucial genotype-dependent differences were also observed. The introgression genotype characterized by an ability to regenerate membranes after re-hydration demonstrated a higher accumulation level of most chloroplast and numerous extra-chloroplast membrane lipid species at the beginning of drought. Furthermore, this genotype also revealed a significant reduction in the accumulation of most chloroplast lipids after re-hydration, compared with the other introgression genotype without the capacity for membrane regeneration. The potential influence of observed lipidomic alterations on a cellular membrane stability and photosynthetic capacity, are discussed. HIGHLIGHTS A higher drought tolerance of grasses could be associated with an earlier lipidome response to a stress signal and with a membrane regeneration after stress cessation accompanied by a turnover of chloroplast lipids.

Analysis of Drought-Induced Proteomic and Metabolomic Changes in Barley (Hordeum vulgare L.) Leaves and Roots Unravels Some Aspects of Biochemical Mechanisms Involved in Drought Tolerance.

In this study, proteomic and metabolomic changes in leaves and roots of two barley (Hordeum vulgare L.) genotypes, with contrasting drought tolerance, subjected to water deficit were investigated. Our two-dimensional electrophoresis (2D-PAGE) combined with matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF and MALDI-TOF/TOF) analyses revealed 121 drought-responsive proteins in leaves and 182 in roots of both genotypes. Many of the identified drought-responsive proteins were associated with processes that are typically severely affected during water deficit, including photosynthesis and carbon metabolism. However, the highest number of identified leaf and root proteins represented general defense mechanisms. In addition, changes in the accumulation of proteins that represent processes formerly unassociated with drought response, e.g., phenylpropanoid metabolism, were also identified. Our tandem gas chromatography - time of flight mass spectrometry (GC/MS TOF) analyses revealed approximately 100 drought-affected low molecular weight compounds representing various metabolite types with amino acids being the most affected metabolite class. We compared the results from proteomic and metabolomic analyses to search for existing relationship between these two levels of molecular organization. We also uncovered organ specificity of the observed changes and revealed differences in the response to water deficit of drought susceptible and tolerant barley lines. Particularly, our results indicated that several of identified proteins and metabolites whose accumulation levels were increased with drought in the analyzed susceptible barley variety revealed elevated constitutive accumulation levels in the drought-resistant line. This may suggest that constitutive biochemical predisposition represents a better drought tolerance mechanism than inducible responses.

Quantitative Trait Loci for Yield and Yield-Related Traits in Spring Barley Populations Derived from Crosses between European and Syrian Cultivars.

In response to climatic changes, breeding programmes should be aimed at creating new cultivars with improved resistance to water scarcity. The objective of this study was to examine the yield potential of barley recombinant inbred lines (RILs) derived from three cross-combinations of European and Syrian spring cultivars, and to identify quantitative trait loci (QTLs) for yield-related traits in these populations. RILs were evaluated in field experiments over a period of three years (2011 to 2013) and genotyped with simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers; a genetic map for each population was constructed and then one consensus map was developed. Biological interpretation of identified QTLs was achieved by reference to Ensembl Plants barley gene space. Twelve regions in the genomes of studied RILs were distinguished after QTL analysis. Most of the QTLs were identified on the 2H chromosome, which was the hotspot region in all three populations. Syrian parental cultivars contributed alleles decreasing traits' values at majority of QTLs for grain weight, grain number, spike length and time to heading, and numerous alleles increasing stem length. The phenomic and molecular approaches distinguished the lines with an acceptable grain yield potential combining desirable features or alleles from their parents, that is, early heading from the Syrian breeding line (Cam/B1/CI08887//CI05761) and short plant stature from the European semidwarf cultivar (Maresi).

Combined mass spectrometric and chromatographic methods for in-depth analysis of phenolic secondary metabolites in barley leaves.

Structural analysis via HPLC-ESI-MSn, UPLC-HESI-MS/MS and NMR reported 152 phenolic secondary metabolites in spring barley seedlings (Hordeum vulgare L.). Flavonoids with various patterns of glycosylation and acylation, as well as hydroxycinnamic acid glycosides, esters and amides, were identified in methanolic extracts from leaves of nine varieties of barley originating from different regions of the world. Hordatines derivatives, flavones acylated directly on the aglycone, and hydroxyferulic acid derivatives deserve special attention. Preparative chromatography enabled characterization of a number of compounds at trace levels with the 6-C-[6″-O-glycosyl]-glycosides and the 6-C-[2″,6″-di-O-glycosides]-glucoside structure of flavones. Derivatives of flavonols, quercetin and isorhamnetin were observed only in Syrian varieties. The ultra performance liquid chromatography profiles of UV-absorbing secondary metabolites were used for chemotaxonomic comparison between nine varieties of barley from different climatic conditions. The hierarchical clustering of bred lines from the Fertile Crescent and European and American varieties indicates a great diversity of chemical phenotypes within barley species.

Comparison of bioactive compounds content in leaf extracts of Passiflora incarnata, P. caerulea and P. alata and in vitro cytotoxic potential on leukemia cell lines

ABSTRACT Passiflora caerulea L., P. alata Curtis and P. incarnata L. (synonym for P. edulis Sims), are the most popular representatives of the Passiflora genus in South America. In recent years, a growing attention is paid to the biological activity and phytochemical profiles of crude extracts from various species of Passiflora in worldwide. The aim of this study was to evaluate and to compare of anti-leukemic activity of the dry crude extracts from leaves of three Passiflora species from greenhouse of Poland in two human acute lymphoblastic leukemia cell lines: CCRF-CEM and its multidrug resistant variant. Two systems of liquid chromatography in order to assessment of phytochemical composition of extracts were applied. Extracts of P. alata and P. incarnata showed the potent inhibitory activity against human acute lymphoblastic leukemia CCRF-CEM, while P. caerulea not showed activity (or activity was poor). Despite similarities in quality phytochemical profile of extracts from P. caerulea and P. incarnata, differences in quantity of chemical compounds may determine their various pharmacological potency. For the activity of P. alata extract the highest content of terpenoids and a lack of flavones C-glycosides are believed to be crucial. Summarizing, the crude extract from P. alata leaves may be considered as a substance for complementary therapy for cancer patients.